Effect of Aloe vera whole leaf extract on short chain fatty acids production by Bacteroides fragilis, Bifidobacterium infantis and Eubacterium limosum.

AIMS: To investigate the effect of Aloe vera whole leaf extract on pure and mixed human gut bacterial cultures by assessing the bacterial growth and changes in the production of short chain fatty acids. METHODS AND RESULTS: Bacteroides fragilis, Bifidobacterium infantis, and Eubacterium limosum were incubated with Aloe vera extracts [0%, 0.5%, 1%, 1.5% and 2%; (w/v)] for 24 and 48 h. Short chain fatty acids production was measured by gas chromatography/mass spectrometry analyses. A significant linear increase in growth response to Aloe vera supplementation was observed at 24 h for each of the bacterial cultures; however, only B. infantis and a mixed bacterial culture showed a significant positive linear dose response in growth at 48 h. In pure bacteria cultures, a significantly enhanced dose response to Aloe vera supplementation was observed in the production of acetic acid by B. infantis at 24 h and of butyric acid by E. limosum at 24 and 48 h. In the mixed bacterial culture, the production of propionic acid was reduced significantly at 24 and 48 h in a dose-dependent fashion, whereas butyric acid production showed a significant linear increase. CONCLUSIONS: The results indicated that Aloe vera possessed bacteriogenic activity in vitro and altered the production of acetic, butyric and propionic acids by micro-organisms selected for the study. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the study suggest that consumption of a dietary supplement, Aloe vera, may alter the production of short chain fatty acids by human intestinal microflora.

Synthesis, spectral analysis, and mutagenicity of 1-, 3-, and 6-nitrobenzo[a]pyrene.

The mutagenic environmental pollutants 1-, 3-, and 6-nitrobenzo[a]pyrene were synthesized. Nitration of 7,8,9,10-tetrahydrobenzo[a]pyrene with sodium nitrate in trifluoroacetic acid and acetic anhydride at ambient temperature gave a mixture of 1-, 3-, and 6-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, which was separated by chromatography. Dehydrogenation of the isolated nitrotetrahydrobenzo[a]pyrenes with 2,3-dichloro-4,5-dicyano-1,6-benzoquinone produced 1-, 3-, and 6-nitrobenzo[a]pyrene in high yield. Comparison of the spectral data of these compounds with those obtained from direct nitration of benzo[a]pyrene confirmed that 1- and 3-nitrobenzo[a]pyrenes are indeed the minor products of the latter reaction. This confirmation also verifies that 1- and 3-nitrobenzo[a]pyrene were the minor nitrated products of benzo[a]pyrene formed in model air atmospheres. The 1-, 3-, and 6-nitrobenzo[a]pyrene were mutagenic in Salmonella typhimurium tester strains TA98 and TA100 in the presence of a mammalian microsomal (S9) activating system. Both 1- and 3-nitrobenzo[a]pyrene, but not 6-nitrobenzo[a]pyrene, were also direct-acting mutagens in these strains. However, only 6-nitrobenzo[a]pyrene exhibited weak mutagenic activity when tested in Chinese hamster ovary cells, while only 3-nitrobenzo[a]pyrene produced a concentration-dependent decrease in cellular survival.

Naphtho and benzo analogues of the kappa opioid agonist trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide.

Further elaboration on the structure-activity relationships in our U-50,488 series has revealed that benzologation of this cyclohexane-1,2-diamine derivative provides compounds which either maintain the interaction with the kappa receptor (e.g. compounds 3a and 5a in the phenylacetamido series) or eliminate the mu receptor mediated analgesia (e.g. compounds 3-6 in the benzamido series). Naphthologation also caused the elimination of mu receptor mediated analgesia (e.g. compounds 17a and 17b).

Evidence for the role of 2-hydroxychromene-2-carboxylate isomerase in the degradation of anthracene by Sphingomonas yanoikuyae B1.

Sphingomonas yanoikuyae B1 is extremely versatile in its catabolic ability. An insertional mutant strain, S. yamoikuyae EK504, which is unable to grow on naphthalene due to the loss of 2-hydroxychromene-2-carboxylate isomerase activity, was utilized to investigate the role of this enzyme in the degradation of anthracene by S. yanoikuyae B1. Although EK504 is unable to grow on anthracene, this strain could transform anthracene to some extent. A metabolite in the degradation of anthracene by EK504 was isolated by high-pressure liquid chromatography (HPLC) and was identified as 6,7-benzocoumarin by UV-visible, gas-chromatographic, HPLC/mass-spectrometric, and 1H nuclear magnetic resonance spectral techniques. The identification of 6,7-benzocoumarin provides direct chemical and genetic evidence for the involvement of nahD in the degradation of anthracene by S. yanoikuyae B1.

Metabolism of anthracene by a Rhodococcus species.

A Rhodococcus sp. isolated from contaminated river sediment was investigated to determine if the isolate could degrade high molecular mass polycyclic aromatic hydrocarbons. The Rhodococcus sp. was able to utilize anthracene (53%), phenanthrene (31%), pyrene (13%), and fluoranthene (5%) as sole source of carbon and energy, but not naphthalene or chrysene. In a study of the degradation of anthracene by a Rhodococcus sp., the identification of ring-fission products indicated at least two ring-cleavage pathways. One results in the production of 6,7-benzocoumarin, previously shown to be produced chemically from the product of meta cleavage of 1,2-dihydroxyanthracene, a pathway which has been well established in Gram-negative bacteria. The second is an ortho cleavage of 1,2-dihydroxyanthracene that produces 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, a dicarboxylic acid ring-fission product. This represents a novel metabolic pathway only identified in Gram-positive bacteria.

Regioselective transformation of ciprofloxacin to N-acetylciprofloxacin by the fungus Mucor ramannianus.

A strain of the saprobic fungus Mucor ramannianus, isolated from a forest, was used to demonstrate the potential for ciprofloxacin biotransformation by zygomycetes in the environment. The fungus carried out the regioselective N-acetylation of ciprofloxacin to a single product, which was purified from culture extracts by high-performance liquid chromatography. The metabolite was identified by mass and nuclear magnetic resonance spectrometry as 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(4-acetyl-1-piperazinyl)-3- quinolinecarboxylic acid.

Extravascular fluid uptake during cardiopulmonary bypass in hypertensive dogs.

We investigated the effects of increases in central venous pressure (CVP) and carotid baroreceptor-induced vasodilation on the rate of extravascular fluid uptake during cardiopulmonary bypass in normotensive and Goldblatt hypertensive dogs. Carotid sinus baroreceptors were selectively perfused to control the level of vasodilation. Central venous pressure was controlled by changing the height of the venous outflow cannula. Extravascular fluid uptake was determined from the rate of change in reservoir volume. After 3 hours of bypass, total fluid accumulation was 56.11 +/- 14.16 mL/kg in normotensive dogs, significantly less than in hypertensive dogs (110.90 +/- 23.20 mL/kg) (p < 0.05). Raising CVP from 1 to 5 mm Hg increased the rate of extravascular fluid uptake in both normotensive (from 0.05 +/- 0.25 to 0.85 +/- 0.22 mL.kg-1.min-1; p < 0.05) and hypertensive dogs (from 0.68 +/- 0.28 to 2.57 +/- 0.46 mL.kg-1.min-1; p < 0.01)). At a constant CVP, baroreceptor-induced vasodilation increased the rate of extravascular fluid uptake in normotensive (from 0.25 +/- .15 to 0.81 +/- .22 mL.kg-1.min-1) and in hypertensive dogs (from 0.84 +/- .12 to 1.72 +/- .32 mL.kg-1.min-1; p < 0.05). Hypertensive dogs were more sensitive to changes in CVP and to baroreceptor-induced vasodilation. The results of this study imply that elevations in CVP or the use of vasodilators may lead to increased extravascular fluid uptake during bypass; this effect may be exacerbated in the hypertensive state.

Evaluation of major active components in St. John's Wort dietary supplements by high-performance liquid chromatography with photodiode array detection and electrospray mass spectrometric confirmation.

A RP-HPLC method with photodiode array detection and LC-electrospray ionization (ESI) MS confirmation was established for the determination of major active components in St. John's Wort dietary supplement capsules. The samples alternatively were extracted with ethanol-acetone (2:3) using a 55 degrees C water-bath shaker or an ambient temperature ultrasonic bath. Extracts were separated by RP-C18 chromatography using a 95-min water-methanol-acetonitrile-trifluoroacetic acid gradient. The major components were identified by photodiode array detection and then confirmed by LC-ESI-MS. The quantification of components was performed using an internal standard (luteolin). This method may serve as a valuable tool for the quality evaluation of St. John's Wort dietary supplement products.

Microbial metabolism of pyrene.

The isolation and identification of pyrene metabolites formed from pyrene by the fungus Cunninghamella elegans is described. C. elegans was incubated with pyrene for 24 h. Six metabolites were isolated by reversed-phase high-performance liquid (HPLC) and thin-layer chromatography (TLC) and characterized by the application of UV absorption, 1H-NMR and mass spectral techniques. C. elegans hydroxylated pyrene predominantly at the 1,6- and 1,8-positions with subsequent glucosylation to form glucoside conjugates of 1-hydroxypyrene, 1,6- and 1,8-dihydroxypyrene. In addition, 1,6- and 1,8-pyrenequinones and 1-hydroxypyrene were identified as metabolites. Experiments with [4-14C]pyrene indicated that over a 24-h period, 41% of pyrene was metabolized to ethyl acetate-soluble metabolites. The glucoside conjugates of 1-hydroxypyrene, 1,6- and 1,8-dihydroxypyrene accounted for 26%, 7% and 14% of the pyrene metabolized, respectively. Pyrenequinones accounted for 22%. The results indicate that the fungus C. elegans metabolized pyrene to non-toxic metabolites (glucoside conjugates) as well as to compounds (pyrenequinones) which have been suggested to be biologically active in higher organisms. In addition, there was no metabolism at the K-region of the molecule which is a major site of enzymatic attack in mammalian systems.

Formation of mammalian metabolites of cyclobenzaprine by the fungus, Cunninghamella elegans.

The fungus, Cunninghamella elegans, was used as a microbial model of mammalian drug metabolism to biotransform a tricyclic antidepressant, cyclobenzaprine. Seventy-five percent of this drug at a concentration of 1 mM was metabolized within 72 h by C. elegans grown on Sabouraud dextrose broth. Milligram amounts of fungal metabolites were isolated by reversed-phase high performance liquid chromatography (HPLC) and their structures were characterized by 1H NMR spectroscopy, mass spectrometry, and UV spectroscopy analyses. The major fungal metabolites of cyclobenzaprine were 2-hydroxycyclobenzaprine (59%), N-desmethylcyclobenzaprine (21%), cyclobenzaprine trans-10,11-dihydrodiol (5%), N-desmethyl-2-hydroxy-cyclobenzaprine (3%), 3-hydroxycyclobenzaprine (3%), and cyclobenzaprine N-oxide (1%). These fungal metabolites were used as standards to investigate the metabolism of cyclobenzaprine by rat liver microsomes. Rat liver microsomes also biotransformed cyclobenzaprine to produce similar metabolites as the fungus. The isotope labeling of 2-hydroxycyclobenzaprine by 18O2 and the trans-configuration of the dihydrodiol suggested that these reactions were catalyzed by cytochrome P-450 monooxygenases in C. elegans. These results also demonstrated that the fungal biotransformation system could be used to predict and synthesize the mammalian drug metabolites.

Stereochemistry and evidence for an arene oxide-NIH shift pathway in the fungal metabolism of naphthalene.

The mechanism of naphthalene oxidation by the filamentous fungus, Cunninghamella elegans is described. C. elegans oxidized naphthalene predominately to trans-1,2-dihydroxy-1,2-dihydroxy-1,2-dihydronaphthalene. A trans configuration was assigned for the dihydrodiol by nuclear magnetic resonance (NMR) spectroscopy at 500 MHz which showed a large coupling constant (J1,2) of 11.0 Hz. Comparison of the circular dichroism spectrum of the fungal trans-1,2-dihydroxy-1,2-dihydronaphthalene to that formed by mammalian enzyme systems indicated that the fungal dihydrodiol contained 76% (+)-(1S,2S)-dihydrodiol as the predominant enantiomer. Other naphthalene metabolites formed by C. elegans were identified as 1-naphthol, 2-naphthol and 4-hydroxy-1-tetralone. Incubation of C. elegans with naphthalene and 18O2 indicated that the trans-1,2-dihydroxy-1,2-dihydronaphthalene contained one atom of molecular oxygen which indicated a monooxygenase catalyzed reaction while similar incubations with naphthalene and H182O indicated that the other oxygen atom in trans-1,2-dihydroxy-1,2-dihydronaphthalene was derived from water. Mass spectral analysis of the acid-catalyzed dehydration products of the dihydrodiol indicated that the naphthalene dihydrodiol forms via the addition of water at the C-2 position of naphthalene-1,2-oxide. Fungal metabolism of [1-2H]naphthalene yielded 1-naphthol which retained 78% of the deuterium. NMR analysis of the deuterated 1-naphthol indicated an NIH shift mechanism in which deuterium migrated from the C-1 position to the C-2 position. The above results indicate that naphthalene-1,2-oxide is an intermediate in the fungal metabolism of naphthalene and that the fungal enzymes are highly stereo-selective in the formation of trans-1,2-dihydroxy-1,2-dihydronaphthalene.

Quantitative determination of dexamethasone in human plasma by stable isotope dilution mass spectrometry.

An analytical method for the quantitation of nanogram to subnanogram amounts of dexamethasone is described. Dexamethasone was isolated from human plasma using a C18-bonded reverse-phase cartridge, purified by subsequent normal-phase HPLC, and the corresponding trimethylsilyl derivative analyzed by gas chromatography-mass spectrometry (GC-MS). The quantitation by isotope-dilution MS was carried out by selected-ion monitoring on the (M + 1)+ ion of the trimethylsilyl derivative of dexamethasone and its stable isotopically labeled diluent, [ 13C6 ,2H3]dexamethasone (681 and 690 m/z, respectively). Methane was used as the GC carrier gas and as the chemical-ionization reagent gas. The sensitivity of the method, judged from the lower limit of detection of the mass spectrometer, was at approximately 100 pg. The inter- and intraassay coefficients of variation (CV) determined at two different concentrations were 3.83 and 3.78% for 2 ng/mL and 2.64 and 1.29% for 5 ng/mL, respectively. Plasma concentration profiles for dexamethasone following a single 1-mg iv and a 2-mg oral dose of dexamethasone administered 24 h apart to two healthy volunteers are presented. The mass fragmentographic method described here is useful for bioavailability and pharmacokinetic studies of the synthetic glucocorticoid.

Biotransformation of vinclozolin by the fungus Cunninghamella elegans.

This study investigated the biotransformation of the dicarboximide fungicide vinclozolin [3-(3,5-dichlorophenyl)-5-methyl-5-vinyl-1,3-oxazolidine-2,4-dione] by the fungus Cunninghamella elegans. Experiments with phenyl-[U-ring-14C]vinclozolin showed that after 96 h incubation, 93% had been transformed to four major metabolites. Metabolites were separated by HPLC and characterized by mass and NMR spectroscopy. Biotransformation occurred predominantly on the oxazolidine-2,4-dione portion of vinclozolin. The metabolites were identified as the 3R- and 3S- isomers of 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide, N-(2-hydroxy-2-methyl-1-oxobuten-3-yl)-3,5-dichlorophenyl-1-carbamic acid, and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide. The enanilide compound has been reported previously as a plant and mammalian metabolite and is implicated to contain antiandrogenic activity. The 3R- and 3S- isomers of 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide are novel metabolites.

Characterization of the antihistamines tripelennamine, methapyrilene, and thenyldiamine and their N-oxides by thermospray mass spectrometry.

This study describes an investigation of the thermospray (TS) mass spectrometric analysis of tripelennamine, methapyrilene, thenyldiamine, and their N-oxide derivatives. These compounds were analyzed by direct injection TS mass spectrometry in the column bypass mode and with 0.1M ammonium acetate/methanol (80:20, v/v) as the mobile phase. Typically, the parent antihistamines produced only [MH]s ions under these conditions. The N-oxides provided strong [MH]s ions and multiple fragment ions. A scheme to explain the fragmentation patterns is proposed.

Determination of amoxicillin in catfish and salmon tissues by liquid chromatography with precolumn formaldehyde derivatization.

A liquid chromatographic (LC) method with fluorescence detection was developed for analysis of amoxicillin in catfish and salmon tissues. The tissue was extracted with phosphate buffer (pH 4.5), followed by trichloroacetic acid (TCA) precipitation of proteins and solid-phase (C18) extraction. Trace amounts of nonpolar interfering substances present after solid-phase extraction were removed by ether liquid-liquid extraction. The extract was reacted with formaldehyde and TCA at 100 degrees C for 30 min. A fluorescent derivative was extracted with ether, concentrated, and analyzed by reversed-phase LC with fluorescence detection. Average recoveries of amoxicillin spiked at 2.5-20 ppb were > 80% for catfish and > 75% for salmon muscle tissue, with coefficients of variation of < 6%. Limits of detection (LOD) and quantitation (LOQ) for catfish tissue were 0.5 and 1.2 ppb, respectively. LOD and LOQ for salmon muscle tissue were 0.8 and 2.0 ppb, respectively.

Identification of metabolites produced from N-phenylpiperazine by Mycobacterium spp.

Mycobacterium sp. 7E1B1W and seven other mycobacterial strains known to degrade hydrocarbons were investigated to determine their ability to metabolize the piperazine ring, a substructure found in many drugs. Cultures were grown at 30 degrees C in tryptic soy broth and dosed with 3.1 mM N-phenylpiperazine hydrochloride; samples were removed at intervals and extracted with ethyl acetate. Two metabolites were purified from each of the extracts by high-performance liquid chromatography; they were identified by mass spectrometry and (1)H nuclear magnetic resonance spectroscopy as N-(2-anilinoethyl)acetamide and N-acetyl-N'-phenylpiperazine. The results show that mycobacteria have the ability to acetylate piperazine rings and cleave carbon-nitrogen bonds.

Changes in nasal tip projection and rotation after septorhinoplasty: a cephalometric analysis.

PURPOSE: This prospective study reports on changes in nasal tip projection and nasal tip rotation before and after septorhinoplasty analyzed cephalometrically. PATIENTS AND METHODS: Forty patients undergoing primary septorhinoplasty were studied prospectively. Lateral cephalometric radiographs taken in the natural head position were obtained before and 6 to 45 months (mean, 17.1) after surgery. In 10 patients, serial radiographs were taken at 6 and 12 months, and in seven patients they were taken at 12 and 24 months after surgery. Nasal tip projection (NTP) was defined as the distance between articulare (Ar) and pronasion (PRN). Nasal tip rotation (NTR) was defined as the change in the angle (N-Ar-PRN) after surgery. A surgical goal to increase, decrease, or maintain NTP and NTR was assigned to each patient before surgery. RESULTS: NTP changed in the desired direction in 16 of 40 patients (40%). NTR changed in the desired direction in 25 of 40 patients (63%). In the patients studied serially, NTP decreased an average 0.7 mm between 6 and 12 months (P = .018), and 0.6 mm between 12 and 24 months (P = .071). CONCLUSIONS: Decreased NTP and NTR were the most easily achieved surgical objectives. Maintaining or increasing NTP is less predictable. Typically, there is a progressive loss of NTP after surgery independent of the surgical goal. Cephalometric analysis is a useful tool to measure changes in NTP and NTR after septorhinoplasty.

Radiation doses of commonly used dental radiographic surveys.

The purpose of this study was to evaluate and to compare the radiation dose associated with commonly used dental radiographic surveys including the following: (1) 20 film full-mouth survey, (2) bite-wing radiographs, (3) panoramic survey supplemented with bite-wing radiographs and (4) a common orthodontic radiographic survey (a lateral cephlometric radiograph supplemented with a panoramic radiograph). The effects of collimation and faster radiographic film speeds on dose were also investigated. The effective doses to selected anatomic sites were calculated from measured absorbed doses with the use of an improved, tissue-equivalent phantom fitted with lithium fluoride thermoluminescent dosimeters. It was demonstrated that converting from round to rectangular collimation reduced the radiation exposure by a factor of four. A panoramic survey supplemented with bite-wing radiographs uses approximately one third of the radiation exposure needed to expose a full-mouth survey made with E-speed film and rectangular collimation.

Naphthalene biodegradation in environmental microcosms: estimates of degradation rates and characterization of metabolites.

Naphthalene biodegradation was investigated in microcosms containing sediment and water collected from three ecosystems which varied in past exposure to anthropogenic and petrogenic chemicals. Mineralization half-lives for naphthalene in microcosms ranged from 2.4 weeks in sediment chronically exposed to petroleum hydrocarbons to 4.4 weeks in sediment from a pristine environment. Microbiological analysis of sediments indicated that hydrocarbon-utilizing microbial populations also varied among ecosystems and were 5 to 12 times greater in sediment after chronic petrogenic chemical exposure than in sediment from an uncontaminated ecosystem. Sediment from an ecosystem exposed to agricultural chemicals had a mineralization half-life of 3.2 weeks for naphthalene and showed about a 30-fold increase in heterotrophic bacterial populations in comparison to uncontaminated sediments, but only a 2- to 3-fold increase in hydrocarbon-degrading bacteria. Analysis of organic solvent-extractable residues from the microcosms by high-pressure liquid chromatography detected polar metabolites which accounted for 1 to 3% of the total radioactivity. Purification of these residues by thin-layer chromatography and further analysis by gas chromatography-mass spectrometry indicated that cis-1,2-dihydroxy-1,2-dihydronaphthalene, 1-naphthol, salicylic acid, and catechol were metabolites of naphthalene. These results provide useful estimates for the rates of naphthalene mineralization in different natural ecosystems and on the degradative pathway for microbial metabolism of naphthalene in freshwater and estuarine environments.

Evidence for an arene oxide-NIH shift pathway in the transformation of naphthalene to 1-naphthol by Bacillus cereus.

Bacillus cereus ATCC 14579 transformed naphthalene predominately to 1-naphthol. Experiments with [14C]naphthalene showed that over a 24 h period, B. cereus oxidized 5.2% of the added naphthalene. 1-Naphthol accounted for approximately 80% of the total metabolites. B. cereus incubated with naphthalene under the presence of 18O2 led to the isolation of 1-naphthol that contained 94% 18O. The metabolism of [1-2H]- and [2-2H]-naphthalene by B. cereus yielded 1-naphthol which retained 95% and 94% deuterium, respectively, as determined by mass spectral analysis. NMR spectroscopic analysis of the deuterated 1-naphthol formed from [1-2H]-naphthalene indicated an NIH shift mechanism in which 19% of the deuterium migrated from the C-1 to the C-2 position. The 18O2 and NIH shift experiments implicate naphthalene-1,2-oxide as an intermediate in the formation of 1-naphthol from naphthalene by B. cereus.