Max L. Deinzer

Quantitative analysis of xanthohumol and related prenylflavonoids in hops and beer by liquid chromatography–tandem mass spectrometry

A method for quantitation of six prenylflavonoids (xanthohumol, isoxanthohumol, desmethylxanthohumol, 6- and 8-prenylnaringenins and 6-geranylnaringenin) in hops and beer by HPLC-tandem mass spectrometry has been developed. The method allows direct analysis of beer and crude methanolic extracts of hops. After HPLC separation, prenylflavonoids were detected by positive ion multiple-reaction monitoring using a triple-quadrupole mass spectrometer equipped with a heated nebulizer--atmospheric pressure chemical ionization interface. The accuracy and precision were evaluated by replicate analyses of (spiked) samples. Thirteen commercial beers were analysed with the method. Isoxanthohumol, formed by isomerization of xanthohumol during the brewing process, was the most abundant flavonoid in hopped beers, ranging from 0.04 to 3.44 mg/l.

Prenylflavonoids from Humulus lupulus

Abstract Five flavonoids were isolated from the resin part of the female inflorescences of Humulus lupulus , together with four known hop flavonoids, i.e. xanthohumol, 2′,4′,6′,4-tetrahydroxy-3′- C -prenylchalcone, iso-xanthohumol and 6-prenylnaringenin. The new hop compounds were identified as 2′,4′,6′,4-tetrahydroxy-3′- C -geranylchalcone, 5′-prenylxanthohumol, 6″,6″-dimethylpyrano (2″,3″: 3′,4′)-2′,4-dihydroxy-6′-methoxychalcone, its hydrate and 8-prenylnaringenin; apart from 8-prenylnaringenin, these are new flavonoids. Their mass fragmentation patterns were studied by mass spectrometry using atmospheric pressure chemical ionization in combination with collision-activated decomposition. Loss of the isoprenoid substituent in the positive ion mode and retro Diels-Alder fission in both the positive and negative ion modes provided useful information on the substitution patterns of the A and B rings. Nine hop varieties were qualitatively and quantatively characterized by HPLC-mass spectrometry. The flavonoid profiles of the samples examined were uniform and proved to be of little value in hop variety identification. Xanthohumol was the principal flavonoid in all samples (80–90% of the total of flavonoids) and was accompanied by minor amounts of the other eight flavonoids in virtually all samples.

In vitro inhibition of human P450 enzymes by prenylated flavonoids from hops, Humulus lupulus

1. Several unique flavonoid compounds have recently been isolated from hops, Humulus lupulus, and their presence has been detected in beer. Their chemical structures are similar to other plant-derived compounds, many present in the human diet, that have been shown to have cancer chemopreventive properties due, in part, to inhibition of cytochrome P450 enzymes that activate carcinogens. Additionally, preliminary studies have shown these flavonoids (at 100 muM) to be inhibitory of P450-mediated activation reactions in a variety of in vitro systems. Thus, the in vitro effects of these phytochemicals on cDNA-expressed human CYP1A1, CYP1B1, CYP1A2, CYP3A4 and CYP2E1 were currently examined by the use of diagnostic substrates and the carcinogen AFB1. 2. At 10 muM, the prenylated chalcone, xanthohumol (XN), almost completely inhibited the 7-ethoxyresorufin O-deethylase (EROD) activity of CYP1A1. At the same concentration, other hop flavonoids decreased the EROD activity by 90.8-27.0%. 3. At 10 muM, XN completely eliminated CYP1B1 EROD activity, whereas the other hop flavonoids showed varying degrees of inhibitory action ranging from 99.3 to 1.8%. 4. In contrast, the most effective inhibitors of CYP1A2 acetanilide 4-hydroxylase activitywere the two prenylated flavonoids, 8-prenylnaringenin (8PN) and isoxanthohumol (IX), which produced > 90% inhibition when added at concentrations of 10 mu M. 5. CYP1A2 metabolism of the carcinogen AFB1 was also inhibited by IX and 8PN as shown by decreased appearance of dihydrodiols and AFM1 as analysed by hplc. IX and 8PN also decreased covalent binding of radiolabelled AFB1 to microsomal protein in a concomitant manner. 6. XN, IX and 8PN, however, were poor inhibitors of CYP2E1 and CYP3A4 as measured by their effect on chorzoxazone hydroxylase and nifedipine oxidase activities respectively. 7. These results suggest that the hop flavonoids are potent and selective inhibitors of human cytochrome P450 and warrant further in vivo investigations.

Prenylated chalcones and flavanones as inducers of quinone reductase in mouse Hepa 1c1c7 cells.

The objective of this study was to determine if prenylchalcones (open C-ring flavonoids) and prenylflavanones from hops and beer are inducers of quinone reductase (QR) in the mouse hepatoma Hepa 1c1c7 cell line. All the prenylchalcones and prenylflavanones tested were found to induce QR but not CYP1A1 in this cell line. In contrast, the synthetic chalcone, chalconaringenin, and the flavanone, naringenin, with no prenyl or geranyl groups, were ineffective in inducing QR. The hop chalcones, xanthohumol and dehydrocycloxanthohumol hydrate, also induced QR in the Ah-receptor-defective mutant cell line, Hepa 1c1c7 bp(r)c1. Thus, the prenylflavonoids represent a new class of monofunctional inducers of QR.

Mass Spectrometric Approaches Using Electrospray Ionization Charge States and Hydrogen-Deuterium Exchange for Determining Protein Structures and Their Conformational Changes

Electrospray ionization (ESI) mass spectrometry (MS) is a powerful analytical tool for elucidating structural details of proteins in solution especially when coupled with amide hydrogen/deuterium (H/D) exchange analysis. ESI charge-state distributions and the envelopes of charges they form from proteins can provide an abundance of information on solution conformations that is not readily available through other biophysical techniques such as near ultraviolet circular dichroism (CD) and tryptophan fluorescence. The most compelling reason for the use of ESI-MS over nuclear magnetic resonance (NMR) for measuring H/D after exchange is that larger proteins and lesser amounts of samples can be studied. In addition, MS can provide structural details on transient or folding intermediates that may not be accessible by CD, fluorescence, and NMR because these techniques measure the average properties of large populations of proteins in solution. Correlations between measured H/D and calculated parameters that are often available from crystallographic data can be used to extend the range of structural details obtained on proteins. Molecular dynamics and energy minimization by simulation techniques such as assisted model building with energy refinement (AMBER) force field can be very useful in providing structural models of proteins that rationalize the experimental H/D exchange results. Charge-state envelopes and H/D exchange information from ESI-MS data used complementarily with NMR and CD data provides the most powerful approach available to understanding the structures and dynamics of proteins in solution.

Protein conformations, interactions, and H/D exchange.

Modern mass spectrometry (MS) is well known for its exquisite sensitivity in probing the covalent structure of macromolecules, and for that reason, it has become the major tool used to identify individual proteins in proteomics studies. This use of MS is now widespread and routine. In addition to this application of MS, a handful of laboratories are developing and using a methodology by which MS can be used to probe protein conformation and dynamics. This application involves using MS to analyze amide hydrogen/deuterium (H/D) content from exchange experiments. Introduced by Linderstøm-Lang in the 1950s, H/D exchange involves using (2)H labeling to probe the rate at which protein backbone amide protons undergo chemical exchange with the protons of water. With the advent of highly sensitive electrospray ionization (ESI)-MS, a powerful new technique for measuring H/D exchange in proteins at unprecedented sensitivity levels also became available. Although it is still not routine, over the past decade the methodology has been developed and successfully applied to study various proteins and it has contributed to an understanding of the functional dynamics of those proteins.

Leaf surface flavonoids of Chrysothamnus

Twenty-six flavonoid aglycones have been identified from eight plants covering three species of Chrysothamnus that were collected in eastern Oregon. The flavonoids were identified by NMR spectroscopy, tandem mass spectrometry and co-TLC with authentic markers. Chrysothamnus nauseosus yielded methyl ethers of apigenin, isoscutellarein, luteolin, kaempferol, herbacetin and quercetin. O-Methylated kaempferol and quercetin derivatives were isolated from the leaf exudate of C. humilis. The flavonoid chemistry of C. viscidiflorus was found different from the other two species by the presence of methyl ethers of quercetin, eriodictyol and taxifolin-3-acetate. Although the flavonoid profiles proved of diagnostic value at the species level, they provided little further evidence in favour of inclusion of Chrysothamnus into Ericameria as proposed earlier on the basis of morphological similarities.

Hydrogen/deuterium exchange and mass spectrometric analysis of a protein containing multiple disulfide bonds: Solution structure of recombinant macrophage colony stimulating factor-beta (rhM-CSFβ)

Studies with the homodimeric recombinant human macrophage colony‐stimulating factor beta (rhM‐CSFβ), show for the first time that a large number (9) of disulfide linkages can be reduced after amide hydrogen/deuterium (H/D) exchange, and the protein digested and analyzed successfully for the isotopic composition by electrospray mass spectrometry. Analysis of amide H/D after exchange‐in shows that in solution the conserved four‐helix bundle of (rhM‐CSFβ) has fast and moderately fast exchangeable sections of amide hydrogens in the αA helix, and mostly slow exchanging sections of amide hydrogens in the αB, αC, and αD helices. Most of the amide hydrogens in the loop between the β1 and β4 sheets exhibited fast or moderately fast exchange, whereas in the amino acid 63–67 loop, located at the interface of the two subunits, the exchange was slow. Solvent accessibility as measured by H/D exchange showed a better correlation with the average depth of amide residues calculated from reported X‐ray crystallographic data for rhM‐CSFα than with the average B‐factor. The rates of H/D exchange in rhM‐CSFβ appear to correlate well with the exposed surface calculated for each amino acid residue in the crystal structure except for the αD helix. Fast hydrogen isotope exchange throughout the segment amino acids 150–221 present in rhM‐CSFβ, but not rhM‐CSFα, provides evidence that the carboxy‐terminal region is unstructured. It is, therefore, proposed that the anomalous behavior of the αD helix is due to interaction of the carboxy‐terminal tail with this helical segment.

Electron monochromator-mass spectrometer instrument for negative ion analysis of electronegative compounds

Electron monochromators designed for the production of low-energy electrons (0-15 eV) with nearly monoenergetic distributions have been available for many decades. The concept of adapting the electron monochromator as an ion source onto mass spectrometers for the purpose of electron capture negative ion-mass spectrometric (ECNI-MS) analyses is only now being realized. Two different analyzers, a quadrupole and a double focusing sector instrument, have recently been retrofitted with electron monochromators to test their utility as analytical instruments for the detection of environmental compounds and chemical agents. Electron energy scans of compounds in these classes reveal unique negative ion resonances, which can be used as an additional analytical dimension of information for compound identification and confirmation. Electron currents of 430 μA at 0.03 eV electron energy are now available from the electron monochromator, which will provide sufficient electron flux to meet modern standards for trace level analyses. The narrowest electron energy spread achieved has been ±0.07 eV (fwhm). The electron monochromator-mass spectrometer (EM-MS) instrument has been interfaced to a gas chromatograph (GC), and this system (GC/EM-MS) was used to record ion chromatograms of mixtures of polychlorinated compounds. Regioselective ion loss, resulting from dissociative electron capture by the parent molecule, is electron-energy dependent and can be monitored with the EM-MS instrument. Finally, positive ion spectra produced with monoenergetic electrons have also been recorded.

Immunomodulatory effects of holothurian triterpene glycosides on mammalian splenocytes determined by mass spectrometric proteome analysis

Spleen is a prime organ in which immuno-stimulation takes place in mammalians. Proteome analysis was used to investigate the elicited effects on mouse splenocytes upon exposure to holothurian triterpene glycosides. Cucumarioside A(2)-2, and Frondoside A, respectively, have been used to in-vitro stimulate primary splenocyte cultures. Differential protein expression was monitored by 2D gel analysis and proteins in spots of interest were identified by MALDI ToF MS and nano LC-ESI Q-ToF MS/MS, respectively. Differential image analysis of gels from control vs. gels from stimulated primary splenocyte cultures showed that approximately thirty protein spots were differentially expressed. Prime examples of differentially expressed proteins are NSFL1 cofactor p47 and hnRNP K (down-regulated), as well as Septin-2, NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, and GRB2-related adaptor protein 2 (up-regulated). Immuno-analytical assays confirmed differential protein expression. Together with results from proliferation and cell adhesion assays, our results show that cellular proliferation is stimulated by holothurian triterpene glycosides. In conclusion, holothurian triterpene glycosides are thought to express their immuno-stimulatory effects by enhancing the natural cellular defense barrier that is necessary to fight pathogens and for which lymphocytes and splenocytes have to be recruited constantly due to limited lifetimes of B-cells and T-cells in the body.