Bioavailability of Sulforaphane Following Ingestion of Glucoraphanin-Rich Broccoli Sprout and Seed Extracts with Active Myrosinase: A Pilot Study of the Effects of Proton Pump Inhibitor Administration.

We examined whether gastric acidity would affect the activity of myrosinase, co-delivered with glucoraphanin (GR), to convert GR to sulforaphane (SF). A broccoli seed and sprout extract (BSE) rich in GR and active myrosinase was delivered before and after participants began taking the anti-acid omeprazole, a potent proton pump inhibitor. Gastric acidity appears to attenuate GR bioavailability, as evidenced by more SF and its metabolites being excreted after participants started taking omeprazole.

Investigation into the use of histone deacetylase inhibitor MS-275 as a topical agent for the prevention and treatment of cutaneous squamous cell carcinoma in an SKH-1 hairless mouse model.

Cutaneous squamous cell carcinomas are a common form of highly mutated keratinocyte skin cancers that are of particular concern in immunocompromised patients. Here we report on the efficacy of topically applied MS-275, a clinically used histone deacetylase inhibitor, for the treatment and management of this disease. At 2 mg/kg, MS-275 significantly decreased tumor burden in an SKH-1 hairless mouse model of UVB radiation-induced skin carcinogenesis.

Sulforaphane Bioavailability from Glucoraphanin-Rich Broccoli: Control by Active Endogenous Myrosinase.

Glucoraphanin from broccoli and its sprouts and seeds is a water soluble and relatively inert precursor of sulforaphane, the reactive isothiocyanate that potently inhibits neoplastic cellular processes and prevents a number of disease states. Sulforaphane is difficult to deliver in an enriched and stable form for purposes of direct human consumption. We have focused upon evaluating the bioavailability of sulforaphane, either by direct administration of glucoraphanin (a glucosinolate, or β-thioglucoside-N-hydroxysulfate), or by co-administering glucoraphanin and the enzyme myrosinase to catalyze its conversion to sulforaphane at economic, reproducible and sustainable yields.

Purification of active myrosinase from plants by aqueous two-phase counter-current chromatography.

Introduction: Myrosinase (thioglucoside glucohydrolase; E.C. 3.

Protection of humans by plant glucosinolates: efficiency of conversion of glucosinolates to isothiocyanates by the gastrointestinal microflora.

Plant-based diets rich in crucifers are effective in preventing cancer and other chronic diseases. Crucifers contain very high concentrations of glucosinolates (GS; β-thioglucoside-N-hydroxysulfates). Although not themselves protective, GS are converted by coexisting myrosinases to bitter isothiocyanates (ITC) which defend plants against predators.

Inactivation of tautomerase activity of macrophage migration inhibitory factor by sulforaphane: a potential biomarker for anti-inflammatory intervention.

Background: Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine with keto-enol tautomerase activity, rises rapidly in response to inflammation and is elevated in many chronic diseases. Isothiocyanates, such as sulforaphane from broccoli, are very potent inactivators of MIF tautomerase activity. A simple rapid method for determining this activity in tissues and body fluids may therefore be valuable for assessing severity of inflammation and efficacy of intervention.

An exceptionally potent inducer of cytoprotective enzymes: elucidation of the structural features that determine inducer potency and reactivity with Keap1.

The Keap1/Nrf2/ARE pathway controls a network of cytoprotective genes that defend against the damaging effects of oxidative and electrophilic stress, and inflammation. Induction of this pathway is a highly effective strategy in combating the risk of cancer and chronic degenerative diseases, including atherosclerosis and neurodegeneration. An acetylenic tricyclic bis(cyano enone) bearing two highly electrophilic Michael acceptors is an extremely potent inducer in cells and in vivo.

Safety, tolerance, and metabolism of broccoli sprout glucosinolates and isothiocyanates: a clinical phase I study.

Broccoli sprouts are widely consumed in many parts of the world. There have been no reported concerns with respect to their tolerance and safety in humans. A formal phase I study of safety, tolerance, and pharmacokinetics appeared justified because these sprouts are being used as vehicles for the delivery of the glucosinolate glucoraphanin and its cognate isothiocyanate sulforaphane [1-isothiocyanato-(4R)-(methylsulfinyl)butane] in clinical trials.

Keap1, the sensor for electrophiles and oxidants that regulates the phase 2 response, is a zinc metalloprotein.

Induction of the phase 2 response, a major cellular reaction to oxidative/electrophile stress depends on a protein triad: actin-tethered Keap1 that binds to Nrf2. Inducers react with Keap1 releasing Nrf2 for nuclear translocation and activation of the antioxidant response element (ARE), which regulates phase 2 genes. The primary sensors for inducers are certain uniquely reactive cysteine thiols of Keap1.

Extremely potent triterpenoid inducers of the phase 2 response: correlations of protection against oxidant and inflammatory stress.

A series of synthetic triterpenoid (TP) analogues of oleanolic acid are powerful inhibitors of cellular inflammatory processes such as the induction by IFN-gamma of inducible nitric oxide synthase (iNOS) and of cyclooxygenase 2 in mouse macrophages. Here, we show that these analogues are also extremely potent inducers of the phase 2 response [e.g.

Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteines of the Keap1 sensor modified by inducers.

Induction of a family of phase 2 genes encoding for proteins that protect against the damage of electrophiles and reactive oxygen intermediates is potentially a major strategy for reducing the risk of cancer and chronic degenerative diseases. Many phase 2 genes are regulated by upstream antioxidant response elements (ARE) that are targets of the leucine zipper transcription factor Nrf2. Under basal conditions, Nrf2 resides mainly in the cytoplasm bound to its cysteine-rich, Kelch domain-containing partner Keap1, which is itself anchored to the actin cytoskeleton and represses Nrf2 activity.

Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants.

Coordinate induction of phase 2 proteins and elevation of glutathione protect cells against the toxic and carcinogenic effects of electrophiles and oxidants. All inducers react covalently with thiols at rates that are closely related to their potencies. Inducers disrupt the cytoplasmic complex between the actin-bound protein Keap1 and the transcription factor Nrf2, thereby releasing Nrf2 to migrate to the nucleus where it activates the antioxidant response element (ARE) of phase 2 genes and accelerates their transcription.

An unusual case of 'uncompetitive activation' by ascorbic acid: purification and kinetic properties of a myrosinase from Raphanus sativus seedlings.

Myrosinase (thioglucoside glucohydrolase; EC 3.2.3.

Unexpected genetic and structural relationships of a long-forgotten flavoenzyme to NAD(P)H:quinone reductase (DT-diaphorase).

A mammalian cytosolic FAD-dependent enzyme that catalyzes the reduction of quinones by N-ribosyl- and N-alkyldihydronicotinamides, but not by NADH, NADPH, or NMNH (reduced nicotinamide mononucleotide), was isolated from bovine kidney more than 30 years ago [S. Liao, J. T.

Comprehensive chromatographic and spectroscopic methods for the separation and identification of intact glucosinolates.

Much effort has been devoted to developing methods for the efficient isolation and identification of glucosinolates. Existing methods for separation involve ion exchange, GLC, and HPLC (mostly after chemical modification by enzymatic sulfate removal and/or silylation). We demonstrate a simple and direct strategy for analyzing the glucosinolate content of plant extracts, made possible by a new combination of widely available techniques: (a) reverse-phase paired-ion chromatography (PIC) of plant extracts, (b) hydrolysis of glucosinolates by myrosinase and quantitation of resulting isothiocyanates by cyclocondensation with 1, 2-benzenedithiol; (c) a novel method for replacing the PIC counterions by ammonium ions, permitting direct bioassay, mass, and 1H NMR spectrometry; (d) mass spectrometric analysis of ammonium salts by negative-ion fast atom bombardment (FAB) to determine m/z of the [M - H]- ion, and by chemical ionization (CI) in ammonia to obtain accurate masses of characteristic fragment ions, principally [R-CN:NH4]+, [R-CH=NOH:H]+ and [R-CH=NOH:NH4]+; and (e) high-resolution 1H NMR spectroscopy of intact glucosinolates.

Chemoprotection against cancer by phase 2 enzyme induction.

Mammalian cells have evolved elaborate mechanisms for protection against the toxic and neoplastic effects of electrophilic metabolites of carcinogens and reactive oxygen species. Phase 2 enzymes (e.g.

Mercurials and dimercaptans: synergism in the induction of chemoprotective enzymes.

The induction of NAD(P)H:quinone reductase (EC 1.6.99.

Chemical and molecular regulation of enzymes that detoxify carcinogens.

Inductions of detoxication (phase 2) enzymes, such as glutathione transferases and NAD(P)H:(quinone-acceptor) oxidoreductase, are a major mechanism for protecting animals and their cells against the toxic and neoplastic effects of carcinogens. These inductions result from enhanced transcription, and they are evoked by diverse chemical agents: oxidizable diphenols and phenylenediamines; Michael reaction acceptors; organic isothiocyanates; other electrophiles--e.g.

Measurement of serum levels of dehydroepiandrosterone sulfate: a comparison of radioimmunoassay and enzymatic analysis.

Dehydroepiandrosterone sulfate (DHEAS) and unconjugated dehydroepiandrosterone (DHEA) are secretory products of the adrenal cortex. Measurement of serum levels of these steroids is of increasing epidemiologic interest, since low serum concentrations of DHEAS or DHEA have been associated with an increased risk of dying of cardiovascular disease or of developing cancer. Radioimmunoassays (RIAs) are the most convenient systems for the measurement of serum DHEAS concentrations in multiple samples.

A convenient, unified scheme for the differential extraction of conjugated and unconjugated serum C19 steroids on Sep-Pak C18-cartridges.

In order to conveniently and rapidly isolate by group both conjugated and unconjugated serum androgens, a scheme has been devised for their differential extraction from commercially available, disposable octadecylsilane cartridges (Sep-Pak C18). Using added radioactive steroid standards and detection of endogenous serum steroids by group-specific enzymatic assays, the quantitative recovery of steroid glucuronides and sulfates in the 47% methanol fraction and of unconjugated steroids in the 100% methanol fraction was observed. Maximum recovery of serum protein-bound steroids (e.

The steroidogenic characteristics of primary testicular cell cultures from adult hypophysectomized rats: enhanced formation of C21 steroids.

To characterize and clarify the time-related pattern of steroidogenesis in primary testicular cultures from adult hypophysectomized rats, we have determined the pattern of C19 and C21 steroids using novel enzymatic assay techniques that rely on highly specific bacterial hydroxysteroid dehydrogenases. Steroids contained in culture media were separated in a standardized high performance liquid chromatography system and the 17 beta-hydroxy- and 17-oxosteroids were quantified by a transydrogenase assay. The individual 3 alpha-, 3 beta-, 17 beta-, and 20 alpha-hydroxysteroids were in turn measured by enzymatic oxidation.