High-performance liquid chromatographic assay for the separation and characterization of metabolites of the potential cytostatic drug oracine

Oracine (I), a potential cytostatic drug, is enzymically converted to a number of metabolites whose formation has been studied in vitro and in vivo. The metabolites were separated by reversed-phase HPLC and characterized by UV spectra. Preparative TLC served for the isolation of the individual metabolites to allow their identification. Two metabolites were identified by Fourier transform NMR as 11-dihydrooracine (II) and a phenolic product (III). Two further metabolites (IV,V) were characterized. Some minor, presumably 11-dihydro metabolites and an 11-oxo metabolite produced in vitro and in vivo were revealed.     

Metabolic products of arachidonic acid in rats

The pattern of eicosanoid metabolites appearing in urine and feces following oral administration of radioactive arachidonic acid was investigated using rats deficient in essential fatty acids. About 70–80% of the radioactivity in the urine during the first day after feeding was adsorbed to XAD-2 resin and he represented eicosanoid metabolites, whereas the rest of the radioactivity was mainly ³H₂O. The eicosanoid metabolites were fractioned into different polarity classes using reverse phase Sep-Pak C₁₈ cartridges. Gas chromatographic analysis of the urinary metabolites following their derivatization into methyl ester-methoxime- -butyl-dimethylsilyl ethers revealed that nearly one-half of the metabolites had ECL values less than 22 and represented metabolites more oxidized than commonly described. Only 30% of the metabolites had ECL values between 26 to 32, corresponding to the values for the metabolites that originate from exogenously infused prostaglandins. A large portion of the eicosanoid metabolites was also excreted with the feces. The isotropic patterns from the reverse phase chromatography indicated that many of the fecal metabolites may be similar to those in urine although some metabolites in feces were not present in urine. Based on the specific radioactivity of the administered arachidonic acid, it appeared that at least 6 to 8 mg of eicosanoid metabolites were excreted through urine and feces within 24 hrs following an oral bolus of 60 mg arachidonic acid. The rapid increase and subsequent decrease in eicosanoid metabolite excretion after oral administration of arachidonate indicates that the dietary intake of polyunsaturated fatty acids may have a more rapid effect upon the endogenous production of eicosanoids than is generally recognized.     

Co-culture of Aspergillus sydowii and Bacillus subtilis induces the production of antibacterial metabolites

The co-culture strategy, which mimics natural ecology by constructing an artificial microbial community, is a useful tool to activate the biosynthetic gene clusters to generate new compounds. However, without optimization of fermentation conditions, the antagonism between the microbes often interferes with the production of secondary metabolites. In this study, the fermentation conditions of co-culture of Aspergillus sydowii and Bacillus subtilis were optimized by response surface methodology to increase the production of active metabolites against Staphylococcus aureus. After optimization, the inhibitory rate of the co-culture extract was 74.62%, which was 29.20% higher than that of the initial conditions. Meanwhile, a total of 15 newly biosynthesized metabolites were detected only in optimized co-culture, occupying 13.2% of all detected metabolites. The structures of the 12 metabolites with high variable importance in projection score were elucidated by the established LC-MS/MS approach integrated with various metabonomic tools. Among them, 7 metabolites were newly induced and the content of other 5 metabolites increased by 1.1–2.4 folds in optimized co-culture. The bioassay of metabolites in co-culture against S. aureus indicated that compounds (?)- (7S)- 10-hydroxysydonic acid, serine sydonate and macrolactin U’ contributed much to the increment of antibacterial activity. This study demonstrated that optimizing the fermentation conditions of co-culture was beneficial to changing the metabolite profile and effective to induce the biosynthesis of active metabolites.     

Some aspects of overproduction of secondary metabolites

Different approaches used to increase production of secondary metabolites and construct overproducing strains of microorganisms are reviewed. Overproduction of secondary metabolites incuudes the physiological control,e.g. feed-back inhibition, carbon and energy source regulation, nitrogen source regulation, phosphate regulation and the effect of autoregulatory compounds. The genetic control of overproduction of secondary metabolites includes mechanisms similar to those controlling the expression of primary metabolism coding genes, although the genes specifying biosynthesis of secondary metabolites and their expression have some particular features. Possible future trends in the study of overproduction of secondary metabolites are discussed. Dedicated to the 70th birthday of Dr. Z. Vanêk     

Determination of the stereochemical composition of the major metabolites of verapamil in dog urine with enantioselective liquid chromatographic techniques

The stereochemical composition of verapamil and seven of its basic-extractable metabolites, isolated from the urine of dogs administered oral racemic verapamil, was determined by HPLC, using an Ultron OVM (ovomucoid) column. One dog was given oral (R)-verapamil alone in order to discriminate the (R)- and (S)-enantiomers of the metabolites. Structure identification of the isolated verapamil metabolites was accomplished using a combination of HPLC-MS and FAB-MS-MS techniques. Six of the urinary verapamil metabolites, including verapamil, were predominantly of the (R)-configuration, whereas one of the metabolites was predominantly in the (S)-form. The remaining isolated metabolite was comprised of approximately equal amounts of the two forms.     

Transformation of Verapamil by Cunninghamella blakesleeana

A filamentous fungus, Cunninghamella blakesleeana AS 3.153, was used as a microbial model of mammalian metabolism to transform verapamil, a calcium channel antagonist. The metabolites of verapamil were separated and assayed by the liquid chromatography-ion trap mass spectrometry method. After 96 h of incubation, nearly 93% of the original drug was metabolized to 23 metabolites. Five major metabolites were isolated by semipreparative high-performance liquid chromatography and were identified by proton nuclear magnetic resonance and electrospray mass spectrometry. Other metabolites were characterized according to their chromatographic behavior and mass spectral data. The major metabolic pathways of verapamil transformation by the fungus were N dealkylation, O demethylation, and sulfate conjugation. The phase I metabolites of verapamil (introduction of a functional group) by C. blakesleeana paralleled those in mammals; therefore, C. blakesleeana could be a useful tool for generating the mammalian phase I metabolites of verapamil.     

Metabolic fate of the heart agent [18F]6-fluorometaraminol

Studies were performed to determine whether [¹⁸F]6-fluorometaraminol (¹⁸F-FMR), a new neuronal heart radiopharmaceutical, is metabolized in vivo and if the metabolites are taken up in heart. Rat, dog, baboon and guinea pig were injected with ¹⁸F-FMR and tissue samples were analyzed for metabolites by HPLC. Liver contained the most metabolites of the tissues studied with 25–90% of the radioactivity present as metabolites at 1 h in all the species studied. While metabolites of ¹⁸F-FMR are found in blood, no significant accumulation of these metabolites is found in heart (⩽0.3%) 1 h after i.v. administration in any species except rat. These studies suggest that ¹⁸F-FMR is a suitable agent for quantitative imaging of the heart by positron emission tomography.     

Complete separation by high performance liquid chromatography of metabolites of arachidonic acid from incubation with human and rabbit platelets

Separations of all major cyclooxygenase and lipoxygenase metabolites of arachidonic acid were obtained by high performance liquid chromatography (HPLC). A C₁₈ reverse-phase column was used in ion suppression mode to separate underivatized metabolites of arachidonic acid isolated from human and rabbit platelets. The metabolites were monitored by measuring radioactivity or ultraviolet light absorption at 192 nm (absorption by double bonds). Comparisons of TLC and HPLC separations demonstrated that the HPLC separation of metabolites of [1-¹⁴C]arachidonic acid was quantitative. HPLC also resolved several minor metabolites that were not detected by scanning of TLC separations.     

Production,partial purification and bioassay of toxic metabolites of three plant pathogenic species ofColletotrichum in Nigeria

Colletotrichum lindemuthianum, C. truncatum andC. graminicola, produced metabolites in culture which induced necrotic lesions on susceptible hosts. The highest production was obtained from Richard's medium under shake incubation. The toxic metabolites of these pathogens fluoresce at 254 nm and 366 nm under ultraviolet light. The crude metabolites of theseColletotrichum species inhibited seed germination at the concentration of 100 µg/ml while the potency of the metabolites decreases with increase in dilution to 0.1 µg/ml. Similarly, the metabolites also inhibited growth of seedlings of hosts to the pathogen at 100 µg/ml and the potency again reduced with increasing dilution to 0.10 µg/ml.     

Identification of quinine metabolites in urine after oral dosing in humans

A gas chromatographic–mass spectrometric method was used to separate quinine and its metabolites present in urine after oral dosing of 300 mg quinine in humans. The technique allowed the separation of quinine and ten metabolites. Four of these metabolites were definitely identified as 3-hydroxyquinine, 2′-quinone, O-desmethylquinine and 10,11-dihydroxydihydroquinine, by comparing their methane chemical ionization mass spectra with those of authentic standards prepared by organic synthesis. Six other metabolites are described for the first time in human urine. From their electron impact and chemical ionization mass spectra, we propose these compounds to be 3-hydroxy-2′-quinone, O-desmethyl-2′-quinone, O-desmethyl-3-hydroxyquinine, O-desmethyl-3-hydroxy-2′-quinone, 10,11-dihydroxydihydro-2′-quinone and 10,11-dihydroxydihydro-O-desmethylquinine. These secondary metabolites probably arose from further biotransformation of the four primary metabolites.     

Identification of new urinary metabolites of trimeprazine in rats by gas chromatography—mass spectrometry

The metabolites of trimeprazine were identified in urine of rats by gas chromatography—mass spectrometry. After the oral administration of trimeprazine, the urinary metabolites were extracted with diethyl ether before or after hydrolysis with β-glucuronidase. The identified metabolites were N-demethyltrimeprazine, 3-hydroxytrimeprazine, N-demethyl-3-hydroxytrimeprazine and trimeprazine sulphoxide.     

Metabolic fate of endogenously synthesized prostaglandin D2 in a human female with mastocytosis

Increased production of prostaglandin D₂ was recently demonstrated in patients with systemic mastocytosis. One female patient investigated with mastocytosis was found to have overproduction of prostaglandin D₂ of such magnitude (150-fold above normal) that it provided the unique opportunity to delineate the metabolic fate of endogenously synthesized prostaglandin D₂. A five percent aliquot of a twenty-four hour urine collection from the patient was extracted, purified by silicic acid chromatography, methylated, and finally subjected to high pressure liquid chromatography. Column fractions collected were derivatized and analyzed by gas chromatography-mass spectrometry. Increased quantities of sixteen urinary metabolites were identified and included a series of metabolites retaining the PGD-ring as well as series of metabolites with a PGF-ring. PGF-ring metabolites were excreted in approximately 4-fold greater relative abundance than PGD-ring metabolites. More than one apparent isomeric form of some PGF-ring metabolites were found. The predominant urinary metabolite was 2,3-dinor-prostaglandin F₂. This study provides evidence that endogenously synthesized prostaglandin D₂ is converted in substantial part to prostaglandin F₂ metabolites in humans.     

Metabolomic fingerprinting of bull spermatozoa for identification of fertility signature metabolites

The objective of the study was to identify the fertility‐associated metabolites in bovine spermatozoa using liquid chromatography‐mass spectrometry (LC‐MS). Six Holstein Friesian crossbred bulls (three high‐fertile and three low‐fertile bulls) were the experimental animals. Sperm proteins were isolated and protein‐normalized samples were processed for metabolite extraction and subjected to LC‐MS/MS analysis. Mass spectrometry data were processed using iMETQ software and metabolites were identified using Human Metabolome DataBase while, Metaboanalyst 4.0 tool was used for statistical and pathway analysis. A total of 3,704 metabolites belonging to various chemical classes were identified in bull spermatozoa. After sorting out exogenous metabolites, 56 metabolites were observed common to both the groups while 44 and 35 metabolites were found unique to high‐ and low‐fertile spermatozoa, respectively. Among the common metabolites, concentrations of 19 metabolites were higher in high‐fertile compared to low‐fertile spermatozoa (fold change > 1.00). Spermatozoa metabolites with variable importance in projections score of more than 1.5 included hypotaurine, d ‐cysteine, selenocystine. In addition, metabolites such as spermine and l ‐cysteine were identified exclusively in high‐fertile spermatozoa. Collectively, the present study established the metabolic profile of bovine spermatozoa and identified the metabolomic differences between spermatozoa from high‐ and low‐fertile bulls. Among the sperm metabolites, hypotaurine, selenocysteine, l ‐malic acid, d ‐cysteine, and chondroitin 4‐sulfate hold the potential to be recognized as fertility‐associated metabolites.     

Mass fragmentographic quantitation of ethotoin and some of its metabolites in human urine

A method for the determination of ethotoin and its p-hydroxylated and dealkylated metabolites in urine has been developed. Ethotoin and the metabolites were extracted from acidified urine with ethyl acetate and silylated before injection into a combined gas chromatograph—mass spectrometer. Four partly identified metabolites were recorded, but their exact quantitation was not possible as pure reference substances were not available.The limit of sensitivity was far below the amounts of ethotoin and of its metabolites found in urine from patients treated with therapeutic dozes of ethotoin.     

Excretion of norsteroids’ phase II metabolites of different origin in human

The urinary phase II metabolites of norsteroids, 19-norandrosterone, 19-noretiocholanolone and 19-norepiandrosterone glucuronide and sulphate, were analyzed in samples collected during the pregnancy, following the administration of norsteroids or the consumption of edible parts of non-castrated pig and in athletes’ samples in which they were found during routine controls. The level of the sulfo- and glucuroconjugated metabolites was precisely determined by GC/HRMS, after selective hydrolysis. The goal was to evaluate whether the fine analysis of the norsteroid conjugates produced and excreted in different conditions would show a pattern that could be linked to their origin. The delta ¹³C values of the metabolites formed following the ingestion of edible parts of non-castrated pig were measured by isotope ratio mass spectrometry. Our results indicated that it is not possible to determine the origin of the urinary metabolites based upon the sole evaluation of the different metabolites and conjugates. The GC/C/IRMS is the only method permitting to distinguish between the exogenous and endogenous origin of the metabolites.     

The role of cytochrome P-450 in the synthesis of polar metabolites of aldosterone by microsomes of male rat liver

Among the tissues of the male rat studied, the largest quantities of the neutral polar metabolites of aldosterone were synthesized by the hepatic microsomal fraction. The polar metabolites of aldosterone were separated by HPLC into six peaks. Three peaks of non-polar (reduced) metabolites were also synthesized. Synthesis of at least four of the neutral polar metabolites was induced by phenobarbital and inhibited by both CO and SKF-525A. The rates of synthesis of these metabolites, which were linear up to 5 minutes, correlated well with the concentration of cytochrome P-450 in the liver microsomes. Addition of aldosterone to the microsomal fraction caused a pronounced type 1 change in the cytochrome P-450 spectrum. The half maximal spectral change (K_s) for aldosterone was calculated to be 8 μM. These experiments indicate that the neutral polar metabolites of aldosterone are produced by cytochrome P-450 dependent hydroxy lations.     

High-performance liquid chromatographic separation and isolation of quinidine and quinine metabolites in rat urine

A procedure for the separation and isolation of the urinary metabolites of quinidine and quinine by reversed-phase high-performance liquid chromatography is described. Nine metabolites of quinidine and eight metabolites of quinine were detected in the urine of male Sprague-Dawley rats after a single dose of quinidine or quinine (50 mg kg^?1). Following extraction from urine, the metabolites were separated on either an analytical or a semi-preparative reversed-phase column by gradient elution. After isolation and derivatization, the metabolites were analyzed by gas chromatography and gas chromatography—mass spectrometry.     

Effects of arrhythmogenic lipid metabolites on the L-type calcium current of diabetic vs. non-diabetic rat hearts

Accumulation of lipid metabolites, such as palmitoylcarnitine and lysophosphatidylcholine, is thought to be a major contributor to the development of cardiac arrhythmias during myocardial ischemia. This arrhythmogenicity is likely due to the effects of these metabolites on various ion channels. Diabetic hearts have been shown to accumulate much higher concentrations of these lipid metabolites during ischemia, which may be an important factor in the enhanced incidence of arrhythmias in diabetic hearts. However, it is not known whether these metabolites have similar effects on the ion channels of diabetic hearts as in non-diabetic hearts. Previous studies on myocytes from non-diabetic hearts have reported either enhancement or inhibition of L-type calcium current (I_Ca) by these lipid metabolites. Thus, it is not clear whether the effects of palmitoylcarnitine and/or lysophosphatidlycholine on I_Ca contribute to the enhanced arrhythmogenicity of diabetic hearts or protect against arrhythmias. We determined the effect of exogenous palmitoylcarnitine and lysophosphatidylcholine on the (I_Ca) in ventricular myocytes from streptozotocin-diabetic and non-diabetic rat hearts under identical conditions. We found that palmitoylcarnitine and lysophosphatidylcholine exhibited a dose-dependent inhibition of I_Ca, which was virtually identical in diabetic and non-diabetic cardiac myocytes. Thus, we conclude that these arrhythmogenic lipid metabolites have similar actions on calcium channels in diabetic and non-diabetic hearts. Therefore, the greater susceptibility of diabetic hearts to arrhythmias during myocardial ischemia is not due to an altered sensitivity of the L-type calcium channels to lipid metabolites, but may be explained, in large part, by the greater accumulation of these metabolites during ischemia.     

Effect of dietary calcium and phosphorus on intestinal calcium absorption and vitamin D metabolism.

To understand better dietary regulation of intestinal calcium absorption, a quantitative assessment of the metabolites in plasma and duodenum of rats given daily doses of radioactive vitamin D₃ and diets differing in calcium and phosphorus content was made. All known vitamin D metabolites were ultimately identified by high-pressure liquid chromatography. In addition to the known metabolites (25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, 1,25-dihydroxyvitamin D₃, 25,26-dihydroxyvitamin D₃, and 1,24,25-trihydroxyvitamin D₃), several new and unidentified metabolites were found. In addition to 1,25-dihydroxyvitamin D₃ and 1,24,25-trihydroxyvitamin D₃, the levels of some of the unknown metabolites could be correlated with intestinal calcium transport. However, whether or not any of these metabolites plays a role in the stimulation of intestinal calcium absorption by low dietary calcium or low dietary phosphorus remains unknown.     

Catechol metabolites of the mycotoxin zearalenone are poor substrates but potent inhibitors of catechol-O-methyltransferase

The mycotoxin zearalenone (ZEN) elicits estrogenic effects and is biotransformed to two catechol metabolites, in analogy to the endogenous steroidal estrogen 17ß-estradiol (E2). Previous studies have shown that the catechol metabolites of ZEN have about the same potency to induce oxidative DNA damage as the catechol metabolites of E2, but are less efficiently converted to their methyl ethers by human hepatic catechol-O-methyltransferase (COMT). Here, we report that the two catechol metabolites of ZEN, i.e. 13-hydroxy-ZEN and 15-hydroxy-ZEN, are not only poor substrates of human COMT but are also able to strongly inhibit the O-methylation of 2-hydroxy-E2, the major catechol metabolite of E2. 15-Hydroxy-ZEN acts as a non-competitive inhibitor and is about ten times more potent than 13-hydroxy-ZEN, which is an uncompetitive inhibitor of COMT. The catechol metabolites of ZEN were also shown to inhibit the O-methylation of 2-hydroxy-E2 by hepatic COMT from mouse, rat, steer and piglet, although to a lesser extent than observed with human COMT. The powerful inhibitory effect of catechol metabolites of ZEN on COMT may have implications for the tumorigenic activity of E2, because catechol metabolites of E2 elicit genotoxic effects, and their impaired O-methylation may increase the tumorigenicity of steroidal estrogens.