Daidzein and genistein glucuronides in vitro are weakly estrogenic and activate human natural killer cells at nutritionally relevant concentrations.
Daidzein and genistein glucuronides (DG and GG), major isoflavone metabolites, may be partly responsible for biological effects of isoflavones, such as estrogen receptor binding and natural killer cell (NK) activation or inhibition. DG and GG were synthesized using 3-methylcholanthrene-induced rat liver microsomes. The Km and Vmax for daidzein and genistein were 9.0 and 7.7 micromol/L, and 0.7 and 1.6 micromol/(mg protein. min), respectively. The absence of ultraviolet absorbance maxima shifts in the presence of sodium acetate confirmed that the synthesized products were 7-O-glucuronides. DG and GG were further purified by a Sephadex LH-20 column. DG and GG competed with the binding of 17beta-(3H) estradiol to estrogen receptors of B6D2F1 mouse uterine cytosol. The concentrations required for 50% displacement of 17beta-(3H) estradiol (CB50) were: 17beta-estradiol, 1.34 nmol/L; diethylstilbestrol, 1.46 nmol/L; daidzein, 1.6 micromol/L; DG, 14.7 micromol/L; genistein, 0.154 micromol/L; GG, 7.27 micromol/L. In human peripheral blood NK cells, genistein at <0.5 micromol/L and DG and GG at 0.1-10 micromol/L enhanced NK cell-mediated K562 cancer cell killing significantly (P < 0.05). At > 0.5 micromol/L, genistein inhibited NK cytotoxicity significantly (P < 0.05). The glucuronides only inhibited NK cytotoxicity at 50 micromol/L. Isoflavones, and especially the isoflavone glucuronides, enhanced activation of NK cells by interleukin-2 (IL-2), additively. At physiological concentrations, DG and GG were weakly estrogenic, and they activated human NK cells in nutritionally relevant concentrations in vitro, probably at a site different from IL-2 action. (+info)
Identification of cis-9,10-methylenehexadecanoic acid in submitochondrial particles of bovine heart.
Submitochondrial particles of bovine heart were hydrolyzed by phospholipase A2 and the products were analyzed by liquid chromatography electrospray ionization-mass spectrometry. We found a fatty acid with a molecular mass of 268 Da and a retention time longer than that of linoleic acid. Next, we synthesized organically cis-9,10-methylenehexadecanoic acid, which has a molecular mass similar to that of the extracted fatty acid, and characterized its high performance liquid chromatography and gas chromatography-mass spectrometry profiles. Using these data we were able to identify endogenous cis-9,10-methylenehexadecanoic acid in rat and human heart and liver tissues that had been hydrolyzed by phospholipase A2. This fatty acid was not detected in tissue extracts that had not been hydrolyzed by phospholipase A2. Similar amounts of cis-9, 10-methylenehexadecanoic acid were measured in tissue extracts after total hydrolysis. These results suggest that cis-9, 10-methylenehexadecanoic acid is a fatty acid component, in the sn-2 position, of phospholipids in some mammalian tissue. (+info)
Metabolites of a tobacco-specific carcinogen in urine from newborns.
BACKGROUND: Cigarette smoking during pregnancy can result in fetal exposure to carcinogens that are transferred from the mother via the placenta, but little information is available on fetal uptake of such compounds. We analyzed samples of the first urine from newborns whose mothers did or did not smoke cigarettes for the presence of metabolites of the potent tobacco-specific transplacental carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). METHODS: The urine was collected and analyzed for two metabolites of NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronide (NNAL-Gluc). Gas chromatography and nitrosamine-selective detection, with confirmation by mass spectrometry, were used in the analyses, which were performed without knowledge of the origin of the urine samples. RESULTS: NNAL-Gluc was detected in 22 (71%) of 31 urine samples from newborns of mothers who smoked; NNAL was detected in four of these 31 urine samples. Neither compound was detected in the 17 urine samples from newborns of mothers who did not smoke. The arithmetic mean level of NNAL plus NNAL-Gluc in the 27 newborns of smokers for which both analytes were quantified was 0.14 (95% confidence interval [CI] = 0.083-0.200) pmol/mL. The levels of NNAL plus NNAL-Gluc in the urine from these babies were statistically significantly higher than those in the urine from newborns of nonsmoking mothers (geometric means = 0.062 [95% CI = 0.035-0.110] and 0.010 [considered as not detected; no confidence interval], respectively; two-sided P<.001). NNAL plus NNAL-Gluc levels in the 18 positive urine samples in which both analytes were quantified ranged from 0.045 to 0.400 pmol/mL, with an arithmetic mean level of 0.20 (95% CI = 0.14-0.26) pmol/mL, about 5%-10% of the levels of these compounds detected in the urine from adult smokers. CONCLUSIONS: Two metabolites of the tobacco-specific transplacental carcinogen NNK can be detected in the urine from newborns of mothers who smoked cigarettes during pregnancy. (+info)
Ethyl glucuronide--a marker of alcohol consumption and a relapse marker with clinical and forensic implications.
Ethyl glucuronide (EtG) is a non-volatile, water-soluble, direct metabolite of ethanol that can be detected in body fluids and hair. We investigated urine and serum samples from three patient groups: (1) 33 in-patients in acute alcohol withdrawal; (2) 30 detoxified in-patients (treated for at least 4 weeks) from a 'motivation station'; and (3) 43 neuro-rehabilitation patients (non-alcoholics; most of them suffering from stroke, traumatic brain injury, Parkinson's disease etc.) using gas chromatography/mass spectrometry (GC/MS) with deuterium-labelled EtG as the internal standard and additionally in the second group of patients using liquid chromatography (LC/MS-MS). We found no correlation between the concentration of EtG in urine at hospitalization and the blood-ethanol concentration (r = 0.17), the time frame of detection (r = 0.5) or the total amount of clomethiazole required for the treatment of withdrawal symptoms (r = 0.28). In four out of 30 in-patients from the 'motivation station'--where neither clinical impression nor routine laboratory findings gave indications of relapse--concentrations of EtG in urine ranged between 4.2 and 196.6 mg/l. EtG concentrations in urine of between 2.89 and 23.49 mg/l were found in seven out of 43 neuro-rehabilitation patients using GC/MS. The GC/MS and the LC/MS-MS results showed a correlation of 0.98 with Pearson's correlation test and 1.0 with Spearman's correlation test. We suggest that EtG is a marker of alcohol consumption that can be detected for an extended time period after the complete elimination of alcohol from the body. When used as a relapse marker with a specific time frame of detection intermediate between short- and long-term markers, EtG fills a clinically as well as forensically important gap. Its specificity and sensitivity exceed those of all other known ethanol markers. (+info)
Metabolism of the antimalarial endoperoxide Ro 42-1611 (arteflene) in the rat: evidence for endoperoxide bioactivation.
Ro 42-1611 (arteflene) is a synthetic endoperoxide antimalarial. The antimalarial activity of endoperoxides is attributed to iron(II)-mediated generation of carbon-centered radicals. An alpha, beta-unsaturated ketone (enone; 4-[2',4' bis(trifluoromethyl)phenyl]-3-buten-2-one), obtained from arteflene by reaction with iron(II), was identified previously as the stable product of a reaction that, by inference, also yields a cyclohexyl radical. The activation of arteflene in vivo has been characterized with particular reference to enone formation. [14C]Arteflene (35 micromol/kg) was given i.v. to anesthetized and cannulated male rats: 42.2 +/- 7.0% (mean +/- S.D., n = 7) of the radiolabel was recovered in bile over 5 h. In the majority of rats, the principal biliary metabolites were 8-hydroxyarteflene glucuronide (14.2 +/- 3. 9% dose, 0-3 h) and the cis and trans isomers of the enone (13.5 +/- 4.6% dose, 0-3 h). In conscious rats, 15.3 +/- 1.6% (mean +/- S.D., n = 8) of the radiolabel was recovered in urine over 24 h. The principal urinary metabolite appeared to be a glycine conjugate of a derivative of the enone. Biliary excretion of the glucuronide, but not of the enones, was inhibited by ketoconazole. 8-Hydroxyarteflene was formed extensively by rat and human liver microsomes but no enone was found. Bioactivation is a major pathway of arteflene's metabolism in the rat. Although the mechanism of in vivo bioactivation is unclear, the reaction is not catalyzed by microsomal cytochrome P-450 enzymes. (+info)
Purification and properties of an alginate lyase from a marine bacterium.
An unidentified pseudomonad isolated by enrichment procedures from decomposing seaweed was grown in defined medium containing sodium alginate as the sole carbon source. The alginate lyase recovered from disrupted bacterial cells was purified by a procedure of (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. From sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis experiments a mol.wt. of about 50 000 was determined. The enzyme was active against both algal and bacterial alginate preparations. Kinetic studies together with analysis of the unsaturated oligouronide products of alginate lyase action indicated the enzyme was specific for guluronic acid-containing regions of the macromolecular substrate. The specificity of the enzyme can be used to give information about the primary composition of alginate samples. (+info)
Structural elucidation of a novel exopolysaccharide produced by a mucoid clinical isolate of Burkholderia cepacia. Characterization of a trisubstituted glucuronic acid residue in a heptasaccharide repeating unit.
The structure of the exopolysaccharide (EPS) produced by a clinical isolate of Burkholderia cepacia isolated from a patient with fibrocystic lung disease has been investigated. By means of methylation analyses, carboxyl reduction, partial depolymerization by fuming HCl and chemical degradations such as Smith degradation, lithiumethylenediamine degradation and beta-elimination, supported by GC/MS and NMR spectroscopic analyses, the repeat unit of the EPS has been identified and was shown to correspond to the acidic branched heptasaccharide with the following structure: [formula: see text]. This partially acetylated acidic polymer, distinguished by the presence of the less usual D-isomer of rhamnose and of a trisubstituted glucuronic acid residue, could represent the main EPS produced by this bacterial species. (+info)
Biotransformation of curcumin through reduction and glucuronidation in mice.
Curcumin, the yellow pigment in turmeric and curry, has antioxidative and anticarcinogenic activities. In this study, we investigated the pharmacokinetic properties of curcumin in mice. After i.p. administration of curcumin (0.1 g/kg) to mice, about 2.25 microg/ml of curcumin appeared in the plasma in the first 15 min. One hour after administration, the levels of curcumin in the intestines, spleen, liver, and kidneys were 177.04, 26.06, 26.90, and 7.51 microg/g, respectively. Only traces (0.41 microg/g) were observed in the brain at 1 h. To clarify the nature of the metabolites of curcumin, the plasma was analyzed by reversed-phase HPLC, and two putative conjugates were observed. Treatment of the plasma with beta-glucuronidase resulted in a decrease in the concentrations of these two putative conjugates and the concomitant appearance of tetrahydrocurcumin (THC) and curcumin, respectively. To investigate the nature of these glucuronide conjugates in vivo, the plasma was analyzed by electrospray. The chemical structures of these metabolites, determined by mass spectrometry/mass spectrometry analysis, suggested that curcumin was first biotransformed to dihydrocurcumin and THC and that these compounds subsequently were converted to monoglucuronide conjugates. Because THC is one of the major metabolites of curcumin, we studied its stability at different pH values. THC was very stable in 0.1 M phosphate buffers of various pH values. Moreover, THC was more stable than curcumin in 0.1 M phosphate buffer, pH 7.2 (37 degrees C). These results, together with previous findings, suggest that curcumin-glucuronoside, dihydrocurcumin-glucuronoside, THC-glucuronoside, and THC are major metabolites of curcumin in vivo. (+info)