Richard B. van Breemen

Human, rat, and mouse metabolism of resveratrol.

AbstractPurpose. Resveratrol, a phenolic phytoalexin occurring in grapes, wine, peanuts, and cranberries, has been reported to have anticarcinogenic, antioxidative, phytoestrogenic, and cardioprotective activities. Because little is known about the metabolism of this potentially important compound, the in vitro and in vivo metabolism of trans-resveratrol were investigated. Methods. The in vitro experiments included incubation with human liver microsomes, human hepatocytes, and rat hepatocytes and the in vivo studies included oral or intraperitoneal administration of resveratrol to rats and mice. Methanol extracts of rat urine, mouse serum, human hepatocytes, rat hepatocytes, and human liver microsomes were analyzed for resveratrol metabolites using reversed-phase high-performance liquid chromatography with on-line ultraviolet-photodiode array detection and mass spectrometric detection (LC-DAD-MS and LC-UV-MS-MS). UV-photodiode array analysis facilitated the identification of cis- and trans-isomers of resveratrol and its metabolites. Negative ion electrospray mass spectrometric analysis provided molecular weight confirmation of resveratrol metabolites and tandem mass spectrometry allowed structural information to be obtained. Results. No resveratrol metabolites were detected in the microsomal incubations, and no phase I metabolites, such as oxidations, reductions, or hydrolyzes, were observed in any samples. However, abundant trans-resveratrol-3-O-glucuronide and trans-resveratrol-3-sulfate were identified in rat urine, mouse serum, and incubations with rat and human hepatocytes. Incubation with β-glucuronidase and sulfatase to release free resveratrol was used to confirm the structures of these conjugates. Only trace amounts of cis-resveratrol were detected, indicating that isomerization was not an important factor in the metabolism and elimination of resveratrol. Conclusion. Our results indicate that trans-resveratrol-3-O-glucuronide and trans-resveratrol-3-sulfate are the most abundant metabolites of resveratrol. Virtually no unconjugated resveratrol was detected in urine or serum samples, which might have implications regarding the significance of in vitro studies that used only unconjugated resveratrol.

Caco-2 cell permeability assays to measure drug absorption

Caco-2 cells are a human colon epithelial cancer cell line used as a model of human intestinal absorption of drugs and other compounds. When cultured as a monolayer, Caco-2 cells differentiate to form tight junctions between cells to serve as a model of paracellular movement of compounds across the monolayer. In addition, Caco-2 cells express transporter proteins, efflux proteins, and Phase II conjugation enzymes to model a variety of transcellular pathways as well as metabolic transformation of test substances. In many respects, the Caco-2 cell monolayer mimics the human intestinal epithelium. One of the functional differences between normal cells and Caco-2 cells is the lack of expression of the cytochrome P450 isozymes and in particular, CYP3A4, which is normally expressed at high levels in the intestine. However, Caco-2 cells may be induced to express higher levels of CYP3A4 by treatment with vitamin D3. Caco-2 cell monolayers are usually cultured on semipermeable plastic supports that may be fitted into the wells of multi-well culture plates. Test compounds are then added to either the apical or basolateral sides of the monolayer. After incubation for various lengths of time, aliquots of the buffer in opposite chambers are removed for the determination of the concentration of test compounds and the computation of the rates of permeability for each compound (called the apparent permeability coefficients). Although radiolabelled compounds were used in the original Caco-2 cells monolayer assays, radiolabelled compounds have been replaced in most laboratories by the use of liquid chromatography-mass spectrometry (LC-MS) and LC-tandem mass spectrometry (LC-MS-MS). Mass spectrometry not only eliminates the need for radiolabelled compounds, but permits the simultaneous measurement of multiple compounds. The measurement of multiple compounds per assay reduces the number of incubations that need to be carried out, thereby increasing the throughput of the experiments. Furthermore, LC-MS and LC-MS-MS add another dimension to Caco-2 assays by facilitating the investigation of the metabolism of compounds by Caco-2 cells.

Tomato sauce supplementation and prostate cancer: lycopene accumulation and modulation of biomarkers of carcinogenesis.

As part of a randomized placebo-controlled study to evaluate the effect of lycopene supplementation on DNA damage in men with prostate cancer, a nonrandomized 5th arm using tomato sauce was included and reported here. Thirty-two patients with localized prostate adenocarcinoma consumed tomato sauce-based pasta dishes for 3 weeks (30 mg of lycopene/day) before their scheduled radical prostatectomy. Prostate tissue was obtained as biopsies at baseline and as resected tissue at the time of the prostatectomy. Serum and prostate lycopene, serum prostate specific antigen (PSA) concentrations, and leukocyte DNA 8-OH-deoxyguanosine/deoxyguanosine (80HdG) were measured at baseline and at the end of the intervention. Cancer cells in paraffin sections of prostate biopsies and postintervention resected tissue were compared for 80HdG staining and for apoptosis. Adherence to the daily consumption of tomato-based entrees was 81.6% of the intended dose, and serum and prostate lycopene concentrations increased 1.97- and 2.92-fold (P< 0.001), respectively. Mean serum PSA concentrations decreased by 17.5% (P< 0.002) and leukocyte 80HdG decreased by 21.3% (P< 0.005) after tomato sauce consumption. Resected tissues from tomato sauce-supplemented patients had 28.3% lower prostate 80HdG compared with the nonstudy control group (P < 0.03). Cancer cell 80HdG staining of Gleason Score-matched resected prostate sections was reduced by 40.5% in mean nuclear density (P < 0.005) and by 36.4% in mean area (P < 0.018) compared with the presupplementation biopsy. Apoptotic index was higher in hyperplastic and neoplastic cells in the resected tissue after supplementation. These data taken as a whole indicate significant uptake of lycopene into prostate tissue and a reduction in DNA damage in both leukocyte and prostate tissue. Whether reduction in DNA. damage to prostate cancer cells is beneficial awaits further research, although reduction in serum PSA concentrations is promising.

Time-resolved laser desorption mass spectrometry. I. Desorption of preformed ions

Abstract A laser desorption time-of-flight (LD-TOF) mass spectrometer has been used to study the time-resolved desorption of nonvolatile organic salts following a laser pulse. An electron beam, pulsed prior to the pulse which draws the ions from the source, can be used to examine the neutral species desorbed by the laser. Experiments on tetraalkylammonium halides indicate desorption rates for neutral decomposition products vastly different from those for intact ions and neutral clusters, so that the method can be used to resolve competing thermal processes.

Safety and efficacy of black cohosh and red clover for the management of vasomotor symptoms: a randomized controlled trial.

Objective: The aim of this study was to evaluate the safety and efficacy of black cohosh and red clover compared with placebo for the relief of menopausal vasomotor symptoms. Methods: This study was a randomized, four-arm, double-blind clinical trial of standardized black cohosh, red clover, placebo, and 0.625 mg conjugated equine estrogens plus 2.5 mg medroxyprogesterone acetate (CEE/MPA; n = 89). Primary outcome measures were reduction in vasomotor symptoms (hot flashes and night sweats) by black cohosh and red clover compared with placebo; secondary outcomes included safety evaluation, reduction of somatic symptoms, relief of sexual dysfunction, and overall improvement in quality of life. Results: Reductions in number of vasomotor symptoms after a 12-month intervention were as follows: black cohosh (34%), red clover (57%), placebo (63%), and CEE/MPA (94%), with only CEE/MPA differing significantly from placebo. Black cohosh and red clover did not significantly reduce the frequency of vasomotor symptoms as compared with placebo. Secondary measures indicated that both botanicals were safe as administered. In general, there were no improvements in other menopausal symptoms. Conclusions: Compared with placebo, black cohosh and red clover did not reduce the number of vasomotor symptoms. Safety monitoring indicated that chemically and biologically standardized extracts of black cohosh and red clover were safe during daily administration for 12 months.

Modification of Keap1 Cysteine Residues by Sulforaphane

Activation of the transcription factor NF-E2-related factor-2 (Nrf2) through modification of Kelch-like ECH-associated protein 1 (Keap1) cysteines, leading to up-regulation of the antioxidant response element (ARE), is an important mechanism of cellular defense against reactive oxygen species and xenobiotic electrophiles. Sulforaphane, occurring in cruciferous vegetables such as broccoli, is a potent natural ARE activator that functions by modifying Keap1 cysteine residues, but there are conflicting in vitro and in vivo data regarding which of these cysteine residues react. Although most biological data indicate that modification of C151 is essential for sulforaphane action, some recent studies using mass spectrometry have failed to identify C151 as a site of Keap1 sulforaphane reaction. We have reconciled these conflicting data using mass spectrometry with a revised sample preparation protocol and confirmed that C151 is indeed among the most readily modified cysteines of Keap1 by sulforaphane. Previous mass spectrometry-based studies used iodoacetamide during sample preparation to derivatize free cysteine sulfhydryl groups causing the loss of sulforaphane from highly reactive and reversible cysteine residues on Keap1 including C151. By omitting iodoacetamide from the protocol and reducing sample preparation time, our mass spectrometry-based studies now confirm previous cell-based studies which showed that sulforaphane reacts with at least four cysteine residues of Keap1 including C151.

Psychosine Accumulates in Membrane Microdomains in the Brain of Krabbe Patients, Disrupting the Raft Architecture

Lipid rafts (LRs) are membrane realms characterized by high concentrations of cholesterol and sphingolipids. Often, they are portrayed as scaffolds on which many different signaling molecules can assemble their cascades. The idea of rafts as scaffolds is garnering significant attention as the consequences of LR disruption have been shown to be manifest in multiple signaling pathways. In this study, LRs in the brain of the twitcher (TWI) mouse, a bona-fide model for infant variants of human globoid cell leukodystrophy or Krabbe disease, were investigated. This mouse has deficient activity of GALC (β-galactosylceramidase) that leads to a progressive accumulation of some galactosyl-sphingolipids in the brain. We hypothesized that the accumulation of psychosine (galactosyl-sphingosine) in the TWI CNS may result in the disruption of rafts in different cell populations such as neurons and oligodendrocytes, both cellular targets during disease. In this communication, we demonstrate that psychosine specifically accumulates in LRs in the TWI brain and sciatic nerve and in samples from brains of human Krabbe patients. It is also shown that this accumulation is accompanied by an increase in cholesterol in these domains and changes in the distribution of the LR markers flotillin-2 and caveolin-1. Finally, we show evidence that this phenomenon may provide a mechanism by which psychosine can exert its known inhibitory effect on protein kinase C. This study provides a previously undescribed biophysical aspect for the mechanism of pathogenesis in Krabbe disease.

Identification of photocatalytic degradation products of diazinon in TiO2 aqueous suspensions using GC/MS/MS and LC/MS with quadrupole time-of-flight mass spectrometry

The photocatalytic degradation of the organophosphorus insecticide diazinon in aqueous suspensions has been studied by using titanium dioxide as a photocatalyst. The degradation of the insecticide was a fast process and included the formation of several intermediates that were identified using GC/ion-trap mass spectrometry with EI or CI in positive and negative ionization mode and HPLC/electrospray-QqTOF mass spectrometry. Since primarily hydroxy derivatives were identified in these aqueous suspensions, the mechanism of degradation was probably based on hydroxyl radical attack. The initial oxidative pathways of the degradation of diazinon involved the substitution of sulfur by oxygen on the P=S bond, cleavage of the pyrimidine ester bond, and oxidation of the isopropyl group. Exact mass measurements of the derivatives allowed the elemental formula of the molecules to be determined confidently. Similarities to the metabolic pathways occurring in living organisms were observed.

Liquid chromatography/mass spectrometry of carotenoids using atmospheric pressure chemical ionization

Carotenoids contained in plant extracts were analyzed using liquid chromatography/positive-and negative-ion atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) with a narrow-bore C 30 reversed-phase high-performance liquid chromatographic (HPLC) column and a gradient solvent system containing methanol-methyl tert-butyl ether-ammonium acetate at a flow rate of 300 μl min -1 . In addition to mass spectrometric detection, photodiode-array UV/visible absorbance detection was used between the HPLC column and mass spectrometer for additional carotenoid characterization. Positive-ion APCI produced protonated molecules and molecular ions for both xanthophylls and, unexpectedly, hydrocarbon carotenes ; and during negative-ion APCI, M -. and [M - H] - ions were observed. In order to investigate the origin of the unexpected [M + H] + ions, positive-ion APCI of β-carotene was investigated using deuterochloroform as the only solvent. Because β-carotene formed primarily deuterated ions, [M + D] + , during APCI in deuterochloroform, the mobile phase was determined to be the source of hydrogen for protonation. The hydroxylated xanthophyll lutein fragmented during positive-ion APCI to eliminate water from the protonated molecule and form the base peak of m/z 551. Using collision-induced dissociation in the ion source, additional fragmentation pathways characteristic of tandem mass spectra of carotenoids were observed such as retro-Diels-Alder fragmentation, [M - 56] +. , for α-carotene and loss of toluene from the molecular ion, [M-92] +. for lutein, α-and β-carotene. The limits of detection for protonated molecules of α-carotene and lutein were approximately 3 and 13 pmol, respectively. In negative-ion APCI, the limits of detection were approximately 3 and 1 pmol for M -1 ions of α-carotene and lutein, respectively.

Cyclooxygenase-2 inhibitors in ginger (Zingiber officinale)

Ginger roots have been used to treat inflammation and have been reported to inhibit cyclooxygenase (COX). Ultrafiltration liquid chromatography mass spectrometry was used to screen a chloroform partition of a methanol extract of ginger roots for COX-2 ligands, and 10-gingerol, 12-gingerol, 8-shogaol, 10-shogaol, 6-gingerdione, 8-gingerdione, 10-gingerdione, 6-dehydro-10-gingerol, 6-paradol, and 8-paradol bound to the enzyme active site. Purified 10-gingerol, 8-shogaol and 10-shogaol inhibited COX-2 with IC(50) values of 32 μM, 17.5 μM and 7.5 μM, respectively. No inhibition of COX-1 was detected. Therefore, 10-gingerol, 8-shogaol and 10-shogaol inhibit COX-2 but not COX-1, which can explain, in part, the anti-inflammatory properties of ginger.