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. We show that following administration of glucoraphanin in a commercially prepared dietary supplement to a small number of human volunteers, the volunteers had equivalent output of sulforaphane metabolites in their urine to that which they produced when given an equimolar dose of glucoraphanin in a simple boiled and lyophilized extract of broccoli sprouts. Furthermore, when either broccoli sprouts or seeds are administered directly to subjects without prior extraction and consequent inactivation of endogenous myrosinase, regardless of the delivery matrix or dose, the sulforaphane in those preparations is 3- to 4-fold more bioavailable than sulforaphane from glucoraphanin delivered without active plant myrosinase. These data expand upon earlier reports of inter- and intra-individual variability, when glucoraphanin was delivered in either teas, juices, or gelatin capsules, and they confirm that a variety of delivery matrices may be equally suitable for glucoraphanin supplementation (e.g. fruit juices, water, or various types of capsules and tablets).     

Degradative acetolactate synthase of Bacillus subtilis: purification and properties.

A degradative acetolactate synthase (acetolactate pyruvate-lyase [carboxylating], EC 4.1.3.18) from Bacillus subtilis has been partially purified and characterized. The synthesis of the enzyme was induced by growth of cells in minimal medium plus isobutyrate or acetate. The enzyme was partially purified by ammonium sulfate fractionation, gel filtration, and hydroxyapatite chromatography. The pH optimum of the purified enzyme was 7.0 in phosphate buffer. When assayed in phosphate buffer (pH 7.0), activity was stimulated by acetate and inhibited by sulfate. When assayed in acetate buffer (pH 5.8), activity was inhibited both by sulfate and phosphate. Michaelis-Menten kinetics was observed when the enzyme was assayed in phosphate buffer (pH 6.0 or 7.0), and inhibition by sulfate was competitive and activation by acetate was noncompetitive. When assayed in acetate buffer (pH 5.8), nonlinear Lineweaver-Burk plots were obtained; inhibition by phosphate appeared to be competitive and that by sulfate was of the mixed type. The approximate molecular weight of the purified enzyme was 250,000 as determined by gel filtration.     

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. However, using sera from four individuals we show that different RIA kits provide quite different estimates of serum DHEAS concentrations. Moreover, these results do not always agree with the serum concentrations determined by an independent chromatographic and enzymatic reference method. The results highlight the need for an independent method of determining DHEAS levels in sera that can provide guidance in selecting an appropriate RIA, and in interpreting the results.     

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. Recombinant murine Keap1 contains 0.9 zinc atoms per monomer as determined by inductively coupled plasma-optical emission spectrometry: its zinc content depends on the metal composition of the overexpression medium. Simultaneous direct measurement of bound zinc using a pyridazoresorcinol chelator and protein thiol groups using 4,4'-dipyridyl disulfide has established that (i) zinc is bound to reactive cysteine thiols of Keap1 and is displaced stoichiometrically by inducers, (ii) with these cysteines mutated to alanine, the affinity for zinc is reduced by nearly 2 orders of magnitude, and (iii) the association constant of Keap1 for zinc is 1.02 (+/-0.19) x 10(11) M(-)(1), consistent with a Zn(2+) metalloprotein. Co(2+) substitution for Zn(2+) yields an optical spectrum consistent with tetrahedral metal coordination. Coincident binding of inducers and release of zinc alters the conformation of Keap1, as shown by a profound decline of its tryptophan fluorescence and depression of fluorescence of a hydrophobicity probe. Thus, regulation of the phase 2 response involves chemical modification of critical cysteine residues of Keap1, whose reactivity is modulated by zinc binding. Keap1 is a zinc-thiol protein endowed with a delicate switch controlled by both metal-binding and thiol reactivity.