Lori Coward

Analysis of plasma isoflavones by reversed-phase HPLC-multiple reaction ion monitoring-mass spectrometry

A HPLC-MS procedure for the rapid, sensitive and specific measurement of the isoflavones, daidzein, dihydrodaidzein, O-desmethylangolensin and genistein, in human plasma has been developed. Synthetic radiolabeled genistein conjugates were used for evaluation of optimum conditions for solid phase extraction. Biochanin A was added to plasma as a recovery marker for isoflavones and phenolphthalein glucuronide and 4-methylumbelliferone sulfate were added to ensure completeness of hydrolysis with beta-glucuronidase/sulfatase. Isoflavones in plasma extracts were separated using an isocratic HPLC method and analyzed by negative ion multiple reaction ion monitoring-mass spectrometry using a heated nebulizer-atmospheric pressure chemical ionization interface. Using plasma samples from four subjects consuming two servings a day of an isolated soy protein beverage for 14 days, the mean plasma genistein and daidzein concentrations were 556 and 345 nM, respectively. Within assay and between assay coefficients of variation for measurement of daidzein and genistein in five aliquots of the same plasma sample were 8.51% and 7.76%, and 5.98% and 6.12%, respectively.

High throughput two‐dimensional blue‐native electrophoresis: A tool for functional proteomics of mitochondria and signaling complexes

The recent upsurge in proteomics research has been facilitated largely by streamlining of two‐dimensional (2‐D) gel technology and the parallel development of facile mass spectrometry for analysis of peptides and proteins. However, application of these technologies to the mitochondrial proteome has been limited due to the considerable complement of hydrophobic membrane proteins in mitochondria, which precipitate during first dimension isoelectric focusing of standard 2‐D gels. In addition, functional information regarding protein:protein interactions is lost during 2‐D gel separation due to denaturing conditions in both gel dimensions. To resolve these issues, 2‐D blue‐native gel electrophoresis was applied to the mitochondrial proteome. In this technique, membrane protein complexes such as those of the respiratory chain are solubilized and resolved in native form in the first dimension. A second dimension sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gel then denatures the complexes and resolves them into their component subunits. Refinements to this technique have yielded the levels of throughput and reproducibility required for proteomics. By coupling to tryptic peptide fingerprinting using matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry, a partial mitochondrial proteome map has been assembled. Applications of this functional mitochondrial proteomics method are discussed.

Nitric Oxide-dependent Generation of Reactive Species in Sickle Cell Disease ACTIN TYROSINE NITRATION INDUCES DEFECTIVE CYTOSKELETAL POLYMERIZATION

The intermittent vascular occlusion occurring in sickle cell disease (SCD) leads to ischemia-reperfusion injury and activation of inflammatory processes including enhanced production of reactive oxygen species and increased expression of inducible nitric-oxide synthase (NOS2). Appreciating that impaired nitric oxide-dependent vascular function and the concomitant formation of oxidizing and nitrating species occur in concert with increased rates of tissue reactive oxygen species production, liver and kidney NOS2 expression, tissue 3-nitrotyrosine (NO2Tyr) formation and apoptosis were evaluated in human SCD tissues and a murine model of SCD. Liver and kidney NOS2 expression and NO2Tyr immunoreactivity were significantly increased in SCD mice and humans, but not in nondiseased tissues. TdT-mediated nick end-label (TUNEL) staining showed apoptotic cells in regions expressing elevated levels of NOS2 and NO2Tyr in all SCD tissues. Gas chromatography mass spectrometry analysis revealed increased plasma protein NO2Tyr content and increased levels of hepatic and renal protein NO2Tyr derivatives in SCD (21.4 ± 2.6 and 37.5 ± 7.8 ng/mg) versus wild type mice (8.2 ± 2.2 and 10 ± 1.2 ng/mg), respectively. Western blot analysis and immunoprecipitation of SCD mouse liver and kidney proteins revealed one principal NO2Tyr-containing protein of 42 kDa, compared with controls. Enzymatic in-gel digestion and MALDI-TOF mass spectrometry identified this nitrated protein as actin. Electrospray ionization and fragment analysis by tandem mass spectrometry revealed that 3 of 15 actin tyrosine residues are nitrated (Tyr91, Tyr198, and Tyr240) at positions that significantly modify actin assembly. Confocal microscopy of SCD human and mouse tissues revealed that nitration led to morphologically distinct disorganization of filamentous actin. In aggregate, we have observed that the hemoglobin point mutation of sickle cell disease that mediates hemoglobin polymerization defects is translated, via inflammatory oxidant reactions, into defective cytoskeletal polymerization.

Genistein, a component of soy, inhibits the expression of the EGF and ErbB2/Neu receptors in the rat dorsolateral prostate.

Epidemiological reports suggest that Asians consuming a diet high in soy have a low incidence of prostate cancer. In animal models, soy and genistein have been demonstrated to suppress the development of prostate cancer. In this study, we investigate the mechanism of action, bioavailability, and potential for toxicity of dietary genistein in a rodent model.

Soy Isoflavonoids and Cancer Prevention

The isoflavonoids in soy, genistein and daidzein, have been proposed to contribute an important part of the anti-cancer effect of soy. Although there have been many interesting studies on the effects of isoflavones on biochemical targets in tissue culture experiments, in most cases the concentrations used by investigators have exceeded 10 μM. However, based on simple pharmacokinetic calculations involving daily intake of isoflavones, absorption from the gut, distribution to peripheral tissues, and excretion, it is unlikely that blood isoflavone concentrations even in high soy consumers could be greater than 1–5 μM. Experiments designed to evaluate these pharmacological principles were carried out in anesthetized rats with indwelling biliary catheters and in human volunteers consuming soy beverages. The data from these experiments indicate that genistein is efficiently absorbed from the gut, taken up by the liver and excreted in the bile as its 7-O-β-glucuronide. Re-infused genistein 7-O-β-glucuronide was also well absorbed from the gut, although this occurred in the distal small intestine. In human subjects fed a soy beverage for a period of two weeks, plasma levels of genistein and daidzein, determined by HPLC-mass spectrometry, ranged from 0.55–0.86 μM, mostly as glucuronide and sulfate conjugates. In summary, genistein is well absorbed from the small intestine and undergoes an enterohepatic circulation. Although the plasma genistein levels achievable with soy food feeding are unlikely to be sufficient to inhibit the growth of mature, established breast cancer cells by chemotherapeutic-like mechanisms, these levels are sufficient to regulate the proliferation of epithelial cells in the breast and thereby may cause a chemopreventive effect.

HPLC-Mass Spectrometry Analysis of Isoflavones:

Abstract The current interest in the role of dietary isoflavonoids, particularly the soy isoflavone genistein, in lowering the risk of several chronic diseases, has led to the need for rapid, sensitive and precise assays for isoflavones and their metabolites in food matrices and in various physiological fluids and tissues. HPLC has the advantage over GC-based methods in that all the conjugated and unconjugated isoflavonoids and their metabolites can be separated and analyzed without the need for derivatization. An important advance in mass spectrometry has been the introduction of effective interfaces between the HPLC and the mass spectrometer, namely the electrospray ionization (ESI) and the heated nebulizer-atmospheric pressure chemical ionization (HN-APCI) interfaces. Because of the isoflavonoid concentrations in fluids such as bile or urine, preliminary extraction, so essential for GC-MS and many other analytical methods, is not necessary. This immediately overcomes the thorny issue of finding an effective solid-phase extraction procedure. Using reversed-phase HPLC-ESI-MS, it is possible to obtain a mass/intensity map of all isoflavonoid metabolites in a single 20 min analysis. Analysis of isoflavonoid conjugates in serum/plasma samples requires initial extraction, but the conjugates can be measured intact either by capillary reversed-phase HPLC-ESI-MS or on regular reversed-phase columns by HPLC-HN-APCI-MS. In both cases, specificity is obtained by causing the parent isoflavonoid molecular ions to undergo collision-induced dissociation to form specific daughter ions in a triple quadrupole MS instrument. When it is only necessary to measure the total isoflavonoids and their metabolites in blood, hydrolysis can be performed directly in serum/plasma samples and isoflavonoids recovered by ether or ethyl acetate solvent extraction. The isoflavone aglucones can be analyzed by HPLC-MS under isocratic solvent conditions, thereby drastically shortening analysis time and opening up prospects for automation. Therefore, HPLC-MS is a technique that is broadly applicable to the major issues in phytoestrogen research.

Metabolism of the isoflavones genistein and biochanin A in human breast cancer cell lines.

There is substantial variation in the growth inhibition of different human breast cancer cell lines by the isoflavones genistein and biochanin A. ZR-75-1 and BT-20 cells are > or = 2- to 4-fold less sensitive to these isoflavones than are MCF-7 cells, whereas T47D cells have a sensitivity similar to that of MCF-7 cells. To determine whether these differences are related to isoflavone metabolism by these cancer cells, each of the cell lines was incubated with [4-(14)C]genistein and [4-(14)C]biochanin A. Metabolites in the cell culture media were identified by radio-HPLC electrospray ionization mass spectrometry. One metabolite of genistein (genistein 7-sulfate) and 2 metabolites of biochanin A (genistein and genistein 7-sulfate) were detected by radio-HPLC. Further analysis by mass spectrometry identified 3 other metabolites, a hydroxylated methylated form of each isoflavone and a biochanin A sulfate. IC50 (the concentration at which the growth rate was halved) values of the breast cancer cell lines did not correlate well with production of genistein 7-sulfate from genistein or with biochanin A sulfate, genistein 7-sulfate, or genistein from biochanin A. However, IC50 values correlated with the production of the hydroxylated and methylated forms of the isoflavones. Only T47D cells produced these metabolites in this study, and only T47D cells had IC50 values similar to those of MCF-7 cells, which also produced the hydroxylated and methylated metabolites. These data suggest that the hydroxylated and methylated metabolites may be the active forms of genistein in human breast cancer cells and emphasize the importance of isoflavone metabolism in the mechanism of action of isoflavones.

A Novel Sulindac Derivative That Does Not Inhibit Cyclooxygenases but Potently Inhibits Colon Tumor Cell Growth and Induces Apoptosis with Antitumor Activity

Nonsteroidal anti-inflammatory drugs such as sulindac have shown promising antineoplastic activity, although toxicity from cyclooxygenase (COX) inhibition and the suppression of prostaglandin synthesis limits their use for chemoprevention. Previous studies have concluded that the mechanism responsible for their antineoplastic activity may be COX independent. To selectively design out the COX inhibitory activity of sulindac sulfide (SS), in silico modeling studies were done that revealed the crucial role of the carboxylate moiety for COX-1 and COX-2 binding. These studies prompted the synthesis of a series of SS derivatives with carboxylate modifications that were screened for tumor cell growth and COX inhibitory activity. A SS amide (SSA) with a N,N-dimethylethyl amine substitution was found to lack COX-1 and COX-2 inhibitory activity, yet potently inhibit the growth of human colon tumor cell lines, HT-29, SW480, and HCT116 with IC50 values of 2 to 5 μmol/L compared with 73 to 85 μmol/L for SS. The mechanism of growth inhibition involved the suppression of DNA synthesis and apoptosis induction. Oral administration of SSA was well-tolerated in mice and generated plasma levels that exceeded its in vitro IC50 for tumor growth inhibition. In the human HT-29 colon tumor xenograft mouse model, SSA significantly inhibited tumor growth at a dosage of 250 mg/kg. Combined treatment of SSA with the chemotherapeutic drug, Camptosar, caused a more sustained suppression of tumor growth compared with Camptosar treatment alone. These results indicate that SSA has potential safety and efficacy advantages for colon cancer chemoprevention as well as utility for treating malignant disease if combined with chemotherapy.

Heterogeneity of O-glycosylation in the hinge region of human IgA1

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was applied to studies of the molecular heterogeneity of desialylated human IgA1 hinge region glycopeptides released with two IgA1 proteases. Typically, the hinge region of an alpha1 chain contains three to five O-linked glycan chains. Variants of the hinge region peptides released from IgA1(Kni) myeloma protein carrying 0, 1, 2, or 3 GalNAc residues were observed in the mass spectra as well as the nonglycosylated peptide. Variable numbers of Gal residues indicated additional heterogeneity in O-glycosylation of IgA1. In the hinge region preparation from normal human serum IgA1, glycopeptides carrying 2, 3, 4, or 5 GalNAc residues with variable numbers of Gal residues were detected. In conclusion, our new approach using the site-specific cleavage with two IgA1 proteases allowed precise and sensitive MALDI-TOF mass spectrometric analysis of O-glycosylation heterogeneity in IgA1 hinge region.

HPLC-Mass Spectrometry of Isoflavonoids in Soy and the American Groundnut, Apios Americana

There is an ever growing interest in the study of the polyphenols. Their importance in the prevention of chronic disease is gradually being unfolded. Examples range from those such as the flavonoid quercitin and the stilbene resveratrol in red wine (Das et al., 1999) to the isoflavonoids daidzein, genistein and glycitein in soy (Barnes, 1998).