The pharmacokinetics of anthocyanins and their metabolites in humans.

BACKGROUND AND PURPOSE: Anthocyanins are phytochemicals with reported vasoactive bioactivity. However, given their instability at neutral pH, they are presumed to undergo significant degradation and subsequent biotransformation. The aim of the present study was to establish the pharmacokinetics of the metabolites of cyanidin-3-glucoside (C3G), a widely consumed dietary phytochemical with potential cardioprotective properties. EXPERIMENTAL APPROACH: A 500 mg oral bolus dose of 6,8,10,3',5'-(13)C5-C3G was fed to eight healthy male participants, followed by a 48 h collection (0, 0.5, 1, 2, 4, 6, 24, 48 h) of blood, urine and faecal samples. Samples were analysed by HPLC-ESI-MS/MS with elimination kinetics established using non-compartmental pharmacokinetic modelling. KEY RESULTS: Seventeen (13)C-labelled compounds were identified in the serum, including (13)C5-C3G, its degradation products, protocatechuic acid (PCA) and phloroglucinaldehyde (PGA), 13 metabolites of PCA and 1 metabolite derived from PGA. The maximal concentrations of the phenolic metabolites (Cmax ) ranged from 10 to 2000 nM, between 2 and 30 h (tmax) post-consumption, with half-lives of elimination observed between 0.5 and 96 h. The major phenolic metabolites identified were hippuric acid and ferulic acid, which peaked in the serum at approximately 16 and 8 h respectively. CONCLUSIONS AND IMPLICATIONS: Anthocyanins are metabolized to a structurally diverse range of metabolites that exhibit dynamic kinetic profiles. Understanding the elimination kinetics of these metabolites is key to the design of future studies examining their utility in dietary interventions or as therapeutics for disease risk reduction.

High-performance liquid chromatographic separation of natural and synthetic desulphoglucosinolates and their chemical validation by UV, NMR and chemical ionisation-MS methods.

Methods are described for the optimised extraction, desulphation and HPLC separation of desulphoglucosinolates. These methods provide rapid separation, identification and quantitative measurements of glucosinolates extracted from Brassica napus L and related crops, of unusual glucosinolates found in crucifer weed species, and also of synthetic alkylglucosinolates. The desulphoglucosinolates used in these studies were either chemically synthesised (at least one example from each major structural class), or purified from various plant sources. Validation of the identities of the desulphoglucosinolates was by comparison of retention times with standards, and by UV, 1H- and 13C-NMR and chemical ionisation MS analysis. A list of useful species, and the specific tissues, from which high concentrations of standards can be extracted is included.

A comparative study on the inhibition of human and bacterial kynureninase by novel bicyclic kynurenine analogues.

A series of novel bicyclic analogues of kynurenine were synthesised as inhibitors of kynureninase. The tryptophan-induced bacterial enzyme from Pseudomonas. fluorescens was compared to the constitutive recombinant human enzyme expressed in a baculovirus/insect cell system, with regard to their inhibition by these compounds. All the compounds studied were found to be simple competitive, reversible inhibitors of kynureninase. It was found that altering the size of the second ring of the inhibitor affected the observed Ki values for both enzymes. The addition of an oxygen atom into the second ring had little effect on binding to the bacterial enzyme but gave a more potent inhibitor of human kynureninase. Of the compounds tested, a naphthyl analogue of desaminokynurenine was found to be the most potent inhibitor for both enzymes with Ki values of 5 and 22 microM for bacterial and human enzyme respectively. This report also describes an alternative system for the expression of recombinant human kynureninase which is more convenient for expression in mammalian cells and produces a relatively greater quantity of enzyme.

Synthesis, analysis and rearrangement of novel unnatural glucosinolates.

As part of a structure activity study to examine the interaction of glucosinolates with leaf surfaces, a number of glucosinolates were synthesised bearing novel side chain functionalities. These included 7-carboxyheptyl, heptyl, and naphthyl side chains. For the carboxyheptyl glucosinolate, a novel intramolecular rearrangement reaction was observed during the final deprotection step, which generated an ester attached to the C-3 of glucose. Studies by 1H NMR spectroscopy showed that the hydrophobic side chain associated with one face of the glucose ring and it was proposed that this was the driving force for the rearrangement. Similar hydrophobic interactions were also observed between the heptyl and naphthyl side chains and the glucose.

Mechanism of 3-methylaspartase probed using deuterium and solvent isotope effects and active-site directed reagents: identification of an essential cysteine residue.

The mechanism of the L-threo-3-methylaspartate ammonia-lyase (EC 4.3.1.2) reaction has been probed using deuterium and solvent isotope effects with three different substrates, (2S,3S)-3-methylaspartic acid, (2S)-aspartic acid and (2S,3R)-3-methylaspartic acid. Each substrate appears to form a covalent adduct with the enzyme through the amination of a dehydroalanine (DehydAla-173) residue. The true substrates are N-protonated and at low pH, the alkylammonium groups are deprotonated internally in a closed solvent-excluded pocket after K+ ion, an essential cofactor, has become bound to the enzyme. At high pH, the amino groups of the substrates are able to react with the dehydroalanine residue prior to K+ ion binding. This property of the system gives rise to complex kinetics at pH 9.0 or greater and causes the formation of dead-end complexes which lack Mg2+ ion, another essential cofactor. The enzyme-substrate adduct is subsequently deaminated in two elimination processes. Hydrazines act as alternative substrates in the reverse reaction direction in the presence of fumaric acid derivatives, but cause irreversible inhibition in their absence. Borohydride and cyanide are not inhibitors. N-Ethylmaleimide also irreversibly inactivates the enzyme and labels residue Cys-361. The inactivation process is enhanced in the presence of cofactor Mg2+ ions and Cys-361 appears to serve as a base for the removal of the C-3 proton from the natural substrate, (2S,3S)-3-methylaspartic acid. The dehydroalanine residue appears to be protected in the resting form of the enzyme by generation of an internal thioether cross-link. The binding of the substrate and K+ ion appear to cause a conformational change which requires hydroxide ion. This is linked to reversal of the thioether protection step and generation of the base for substrate deprotonation at C-3. The deamination reaction displays high reverse reaction commitments and independent evidence from primary deuterium isotope effect data indicates that a thiolate acts as the base for deprotonation at C-3.

The 3-methylaspartase reaction probed using 2H- and 15N-isotope effects for three substrates: a flip from a concerted to a carbocationic amino-enzyme elimination mechanism upon changing the C-3 stereochemistry in the substrate from R to S.

The mechanisms of the elimination of ammonia from (2S,3S)-3-methylaspartic acid, (2S)-aspartic acid and (2S,3R)-3-methylaspartic acid, catalysed by the enzyme L-threo-3-methylaspartase ammonia-lyase (EC 4.3.1.2) have been probed using 15N-isotope effects. The 15N-isotope effects for V/K for both (2S,3S)-3-methylaspartic acid and aspartic acid are 1.0246 +/- 0.0013 and 1.0390 +/- 0.0031, respectively. The natural substrate, (2S,3S)-3-methylaspartic acid, is eliminated in a concerted fashion such that the C(beta)-H and C(alpha)-N bonds are cleaved in the same transition state. (2S)-Aspartic acid appears to follow the same mechanistic pathway, but deprotonation of the conjugate acid of the base for C-3 is kinetically important and influences the extent of 15N-fractionation. (2S,3R)-3-Methylaspartic acid is deaminated via a stepwise carbocationic mechanism. Here we elaborate on the proposed model for the mechanism of methylaspartase and propose that a change in stereochemistry of the substrate induces a change in the mechanism of ammonia elimination.

Synthesis of 13C labelled daidzein and formononetin.

Efficient methods are described for the synthesis of daidzein and formononetin labelled with a single 13C atom at the 4-position, to prepare material for metabolic studies.

Studies on the mechanism of myrosinase. Investigation of the effect of glycosyl acceptors on enzyme activity.

Myrosinase (EC 3.2.3.1) is the beta-thioglucosidase enzyme responsible for the hydrolysis of glucosinolates, a group of naturally occurring plant metabolites. The enzyme catalyzes the hydrolysis of these S-glucosides to give D-glucose and an aglycone fragment, which then rearranges to give sulfate and an isothiocyanate. As part of ongoing mechanistic studies on myrosinase, the ability of the enzyme to catalyze transglycosylation reactions has been examined. Enzyme activity and stability were both decreased in the presence of various organic solvents, including simple alcohols, but not sufficiently to prevent reaction taking place. However, in contrast to most other beta-glycosidases, myrosinase did not catalyze transglycosylation reactions either with the alcohols or other suitable glycosyl acceptors. Although a wide range of potential acceptors were investigated, none proved to be effective. Even when appropriately charged side chains were included in the acceptor molecule to mimic the sulfonic acid in the glucosinolate structure, transglycosylation did not take place. The putative enzyme-glycosyl intermediate therefore appears to be unavailable for reaction, possibly because D-glucose is the first product released from the enzyme. The transition state analogue, glucono-delta-lactone, a potent competitive inhibitor of beta-glucosidase, was found to be a poor noncompetitive inhibitor of myrosinase. Myrosinase is specifically activated by ascorbic acid, and it is proposed that the inhibitor is binding at this alternative site.

Cloning, sequencing, and expression in Escherichia coli of the Clostridium tetanomorphum gene encoding beta-methylaspartase and characterization of the recombinant protein.

The gene encoding methylaspartase (EC 4.3.1.2) from Clostridium tetranomorphum has been cloned, sequenced, and expressed in Escherichia coli. The open reading frame (ORF) codes for a polypeptide of 413 amino acid residues (M(r) 45,539) of which seven are cysteine residues. The size of the ORF indicates that methylaspartase is a homodimer rather than an (AB)2 tetramer. The deduced primary structure of the protein shows no homology to enzymes that catalyze similar reactions or, indeed, any convincing homology with any other characterized protein. The recombinant protein is identical to the enzyme isolated directly from C. tetanomorphum as determined by several criteria. The enzyme is obtained in a highly active form (approximately 70% of the activity of the natural enzyme) and migrates as a single band (M(r) 49,000) in SDS-polyacrylamide gels. The kinetic parameters for the deamination of (2S,3S)-3-methylaspartic acid by the natural and recombinant proteins are very similar, and the proteins display identical potassium ion-dependent primary deuterium isotope effects for V and V/K when (2S,3S)-3-methylaspartic acid is employed as the substrate. In accord with the activity of the natural enzyme, the recombinant protein is able to catalyze the slow formation of (2S,3R)-3-methylaspartic acid, the L-erythro-epimer of the natural substrate, from mesaconic acid and ammonia. Earlier work in which the cysteine residues in the protein were labeled with N-ethylmaleimide had indicated that there were eight cysteine residues per protein monomer. One cysteine residue was protected by substrate. Here evidence is forwarded to suggest that the residue that was protected by the substrate is not a cysteine residue but the translation product of a serine codon. Kinetic data indicate that this serine residue may be modified in the active enzyme. The implications of these findings on the mechanism of catalysis are discussed within the context of a few emerging mode of action for methylaspartate ammonia-lyase.

Mechanism of C-3 hydrogen exchange and the elimination of ammonia in the 3-methylaspartate ammonia-lyase reaction.

The enzyme 3-methylaspartate ammonia-lyase (EC 4.3.1.2) catalyzes the exchange of the C-3 hydrogen of the substrate, (2S,3S)-3-methylaspartic acid, with solvent hydrogen. The mechanism of the exchange reaction was probed using (2S,3S)-3-methylaspartic acid and its C-3-deuteriated isotopomer. Incubations conducted in tritiated water allowed the rate of protium or deuterium wash-out from the substrates to be measured as tritium wash-in. The primary deuterium isotope effects for the exchange under essentially Vmax conditions ( [S] much greater than Km) were 1.6, 1.5, and 1.5 at pH 9.0, 7.6, and 6.5. The deamination reaction, measured spectrophotometrically on the same incubations, showed isotope effects of 1.7, 1.6, and 1.4 at pH 9.0, 7.6, and 6.5, in agreement with the values of DV and D(V/K) reported previously [Botting, N.P., Akhtar, M., Cohen, M.A., & Gani, D. (1988) Biochemistry 27, 2956-2959]. The ratio of the rate of exchange to the rate of deamination, however, varied widely with pH. Together with the identical values of the primary isotope effects for the two reactions, this result indicates that the partition between reaction pathways occurs after the slowest steps in the common part of the reaction coordinate pathway, almost certainly after the cleavage of the C-N bond at the level of the enzyme-ammonia-mesaconic acid complex, and not at the putative carbanion level as was previously suggested. The enzyme requires both K+ and Mg2+ ions for activity, although ammonium ion is also able to bind in the K+ site and act as an activator. Variation of the metal ion concentration alters the magnitude of the primary deuterium isotope effects. The variation of potassium ion concentration causes the most marked changes: at 1.6 mM K+, DV and D(V/K) are 1.7, whereas at 50 mM K+, DV and D(V/K) are reduced to 1.0. The isotope effects are also reduced at low K+ concentration due to the emergence of a slow-acting high K+ affinity monopotassium form of the enzyme. The binding order and role of the metal ion cofactors and their influence in determining the formal mechanism of the reaction is discussed, and the failure of previous workers to observe primary deuterium isotope effects for the deamination process is explained. The product desorption order was tested by product inhibition, alternative product inhibition, and isotope exchange experiments. Ammonia and mesaconic acid debind in a random fashion.(ABSTRACT TRUNCATED AT 400 WORDS)

Primary deuterium isotope effects for the 3-methylaspartase-catalyzed deamination of (2S)-aspartic acid, (2S,3S)-3-methylaspartic acid, and (2S,3S)-3-ethylaspartic acid.

3-Methylaspartate ammonia-lyase catalyzes the deamination of (2S)-aspartic acid 137 times more slowly than the deamination of (2S,3S)-3-methylaspartic acid but catalyzes the amination of fumaric acid 1.8 times faster than the amination of mesaconic acid [Botting, N.P., Akhtar, M., Cohen, M. A., & Gani, D. (1988) Biochemistry (preceding paper in this issue)]. In order to understand the mechanistic basis for these observations, the deamination reaction was examined kinetically with (2S)-aspartic acid, (2S,3S)-3-methylaspartic acid, (2S,3S)-3-ethylaspartic acid, and the corresponding C-3-deuteriated isotopomers. Comparison of the double-reciprocal plots of the initial reaction velocities for each of the three pairs of substrates revealed that the magnitude of the primary isotope effect on both Vmax and V/K varied with the substituent at C-3 of the substrate. 3-Methylaspartic acid showed the largest isotope effect (1.7 on Vmax and V/K), 3-ethylaspartic acid showed a smaller isotope effect (1.2 on Vmax and V/K), and aspartic acid showed no primary isotope effect at all. These results, which are inconsistent with earlier reports that there is no primary isotope effect for 3-methylaspartic acid [Bright, H. J. (1964) J. Biol. Chem. 239, 2307], suggest that for both 3-methylaspartic acid and 3-ethylaspartic acid elimination occurs via a predominantly concerted mechanism whereas for aspartic acid an E1cb mechanism prevails.(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate specificity of the 3-methylaspartate ammonia-lyase reaction: observation of differential relative reaction rates for substrate-product pairs.

A range of substituted fumaric and aspartic acid substrates for the enzyme 3-methylaspartate ammonia-lyase (EC 4.3.1.2) have been synthesized and used to study the kinetics of the catalyzed reaction in both the forward (deamination) and reverse (conjugative amination) reaction directions. The rates of amination for all of the alpha, beta-unsaturated substrates studied (bearing substituents the size of an ethyl group or smaller) were similar under [s] much greater than KM conditions although KM values for the substrates varied by a factor of 25. The rates of deamination for the corresponding 3-substituted amino acid substrates varied widely with structure under [s] much greater than KM conditions, and thus for substrate-product pairs the ratio for V(forward)/V(reverse) also varied. These differential reaction rates indicate that there is a step in the deamination direction that is especially sensitive to the size of the 3-substituent of the substrate and that a relatively large group (methyl to ethyl in size) is required for binding in order to reduce the activation energy for this step. Given that it is proposed that the enzyme operates via an E1cb-type mechanism where C-N bond cleavage is rate limiting, it is likely that binding of the C-3 substituent of aspartic acid substrates affects the alignment of the nascent carbanion with the C-N bond for elimination.