Heart-type fatty acid-binding protein (H-FABP) and highly sensitive troponin T (hsTnT) as markers of myocardial injury and cardiovascular events in elective percutaneous coronary intervention (PCI).

BACKGROUND/INTRODUCTION: Type 4a myocardial infarction (MI) occurs when myocardial injury is combined with either symptoms suggestive of myocardial ischaemia, new left bundle branch block, angiographic loss of patency of a major artery or imaging suggestive of new loss of myocardium. Myocardial injury is defined as a rise of >5 x 99th upper reference limit (URL) of 14 ng/l (i.e. >70 ng/l) for highly sensitive troponin T (hsTnT) at 6 h if hsTnT was normal at baseline or >20% rise from 0 to 6 h if hsTnT was >14 ng/l at baseline. AIM: To assess the prognostic value of biomarkers of myocardial injury following elective percutaneous coronary intervention (PCI). DESIGN: A cohort of 209 patients were included of whom 144 (68.9%) were male, mean age was 68.8 years, 28 (13.4%) were smokers, 31 (14.8%) were diabetic, 199 (95.2%) had hypercholesterolaemia and 138 (66.0%) had hypertension. METHODS: We evaluated hsTnT, heart-type fatty acid-binding protein (H-FABP), troponin I (TnI), creatine kinase MB type (CKMB), myoglobin, glycogen phosphorylase BB (GPBB) and carbonic anhydrase III (CA III) at 0, 4, 6 and 24 h following elective PCI. Patients were followed up at 1 year to assess for major adverse clinical events (MACE). RESULTS: Myocardial injury was observed in 37 (17.7%) patients. Median hsTnT/H-FABP at 4 h were most predictive. MACE was noted in 6 (2.9%) patients, 3 had type 4a MI post PCI, P = 0.036. DISCUSSION/CONCLUSIONS: Median 4 h hsTnT/H-FABP were most predictive of myocardial injury following PCI. H-FABP and hsTnT were predictive of MACE.

Vinyl sulfones in solid-phase synthesis: preparation of 4,5,6,7-tetrahydroisoindole derivatives.

The preparation of functionalized 4,5,6,7-tetrahydroisoindole via a traceless solid-phase sulfone linker strategy is described. Thermolytic extrusion of SO(2) from polymer-bound 3-(phenylsulfonyl)-3-sulfolene (7) generated polymer-bound 2-(phenylsulfonyl)-1,3-butadiene (9) in situ which underwent Diels--Alder cycloaddition with various dienophiles to furnish vinyl sulfone resins 10-14. To complete a traceless linker cleavage strategy, (p-tolysulfonyl)methyl isocyanide or ethyl isocyanoacetate was employed to react with the vinyl sulfone moiety to liberate functionalized 4,5,6,7-tetrahydroisoindole products from the resin. Using this chemistry, nine tetrahydroisoindole derivatives (6, 15-22) were prepared in 32-41% overall yields from polystyrene/divinylbenzene sulfinate 1.

Immunoreactivity of organic mimeotopes of the E2 component of pyruvate dehydrogenase: connecting xenobiotics with primary biliary cirrhosis.

In primary biliary cirrhosis (PBC), the major autoepitope recognized by both T and B cells is the inner lipoyl domain of the E2 component of pyruvate dehydrogenase. To address the hypothesis that PBC is induced by xenobiotic exposure, we took advantage of ab initio quantum chemistry and synthesized the inner lipoyl domain of E2 component of pyruvate dehydrogenase, replacing the lipoic acid moiety with synthetic structures designed to mimic a xenobiotically modified lipoyl hapten, and we quantitated the reactivity of these structures with sera from PBC patients. Interestingly, antimitochondrial Abs from all seropositive patients with PBC, but no controls, reacted against 3 of the 18 organic modified autoepitopes significantly better than to the native domain. By structural analysis, the features that correlated with autoantibody binding included synthetic domain peptides with a halide or methyl halide in the meta or para position containing no strong hydrogen bond accepting groups on the phenyl ring of the lysine substituents, and synthetic domain peptides with a relatively low rotation barrier about the linkage bond. Many chemicals including pharmaceuticals and household detergents have the potential to form such halogenated derivatives as metabolites. These data reflect the first time that an organic compound has been shown to serve as a mimeotope for an autoantigen and further provide evidence for a potential mechanism by which environmental organic compounds may cause PBC.

Cationic lipid polymerization as a novel approach for constructing new DNA delivery agents.

In vivo gene delivery mediated by cationic lipids is often compromised by aggregation due to complexation with proteins in the blood. To improve the stability of cationic lipid-DNA complexes, the present study aimed to develop a novel approach in which a poly(cationic lipid) (PCL) is utilized to form stable cationic polyplexes for gene transfection. Hydrogenation of the acrylamide analogue of betaAE-DMRI, the polymerizable precursor of PCL, provided a monomeric lipid derivative (MHL) which was used for direct comparison of corresponding lipoplex stability, toxicity, and transfection activity. Various formulations of cationic liposomes, such as MHL, MHL-cholesterol (Chol), PCL, PCL-Chol, DOTAP-Chol, and commercially available lipofectamine were generated and examined in this study. The new poly(cationic lipid) did not display any significant toxicity to rat hepatocytes or Hep G2 cells as indicated by an LDH leakage assay. Furthermore, PCL was significantly less toxic than MHL, DOTAP-Chol or lipofectamine. Suspensions of PCL were resistant to aggregation even after 24 h of exposure to solutions containing 50 and 100% fetal bovine serum (FBS). In contrast, suspensions of lipofectamine extensively aggregated after 24 h of exposure to 50% FBS. To examine the influence of lipid polymerization on gene transfer activity, liposome-mediated transfections of a luciferase vector (pGL3) were performed in Hep G2 and Alexander cell lines. The luciferase activity of the PCL formulations in Hep G2 cells were similar to those of the MHL, DOTAP-Chol and lipofectamine formulations, demonstrating that lipid polymerization does not compromise transfection activity. In comparison to the monomeric precursor MHL and to the industry transfection standards DOTAP and lipofectamine, the novel poly(cationic lipid) exhibited the lowest cytotoxicity, was the most resistant to serum-induced aggregation and had comparable transfection activity when coformulated with cholesterol. This novel polymerization approach for the development of stable and active polyplexes may prove a valuable alternative for in vivo gene delivery.

Synthesis and reactions of some heterocyclic azacyanines.

The one-step reaction of some amino-substituted heterocycles with diiodomethane to give azacyanines is reported. This useful reaction is of wider application than initially reported and includes the synthesis of new substituted pyrido-, isoquino-, benzimadazo-, and benzothiazoazacyanines 7. Furthermore, treatment of these azacyanines with base generally affects a facile opening of the dihydrotriazinium ring resulting in the formation of new heterocycles 10, 11, and 12, which would be difficult to prepare by other means. This reaction takes an additional direction in the case of halo-substituted azacyanines 7b/c/d where treatment with base gives rise to new interesting derivatives of dipyridotriazines 14b/c/d.

Preparation of a 990-member chemical compound library of hydantoin- and isoxazoline-containing heterocycles using multipin technology.

The development of a useful chemistry for the construction of polyfunctional heterocycles--first through solution and solid phase (resins) and then library production via SynPhase crowns--is reported. Bead-based synthetic work was done on Merrifield resin where treatment with benzylamine in the presence of DBU followed by reaction with 4-chloromethylbenzoyl chloride afforded amide-linked resin 9. Finally, TFA.NH2-polystyrene macro crowns were derivatized with 4-(hydroxymethyl)benzoic acid to afford pin 14 which was coupled with Boc-protected amino acid 2 in the presence of DIC to deliver pin 15. Deprotection and reaction with phenyl isocyanate afforded urea functionalized pin 17 which underwent 1,3-dipolar cycloaddition reaction to give pin 19. Finally, compound 20 was obtained with moderate diastereoselectivity (20:21::8:1) by the reaction of pin 19 with a catalytic amount of Et3N.

Novel CFTR chloride channel activators identified by screening of combinatorial libraries based on flavone and benzoquinolizinium lead compounds.

The flavonoid genistein and the benzo[c]quinolizinium MPB-07 have been shown to activate the cystic fibrosis transmembrane conductance regulator (CFTR), the protein that is defective in cystic fibrosis. Lead-based combinatorial and parallel synthesis yielded 223 flavonoid, quinolizinium, and related heterocyclic compounds. The compounds were screened for their ability to activate CFTR at 50 microm concentration by measurement of the kinetics of iodide influx in Fisher rat thyroid cells expressing wild-type or G551D CFTR together with the green fluorescent protein-based halide indicator YFP-H148Q. Duplicate screenings revealed that 204 compounds did not significantly affect CFTR function. Compounds of the 7,8-benzoflavone class, which are structurally intermediate between flavones and benzo[c]quinoliziniums, were effective CFTR activators with the most potent being 2-(4-pyridinium)benzo[h]4H-chromen-4-one bisulfate (UCcf-029). Compounds of the novel structural class of fused pyrazolo heterocycles were also strong CFTR activators with the most potent being 3-(3-butynyl)-5-methoxy-1-phenylpyrazole-4-carbaldehyde (UCcf-180). A CFTR inhibitor was also identified. The active compounds did not induce iodide influx in null cells deficient in CFTR. Short-circuit current measurements showed that the CFTR activators identified by screening induced strong anion currents in the transfected cell monolayers grown on porous supports. Compared with genistein, the most active compounds had up to 10 times greater potency in activating wild-type and/or G551D-CFTR. The activators had low cellular toxicity and did not elevate cellular cAMP concentration or inhibit phosphatase activity, suggesting that CFTR activation may involve a direct interaction. These results establish an efficient screening procedure to identify CFTR activators and inhibitors and have identified 7,8-benzoflavones and pyrazolo derivatives as novel classes of CFTR activators.

Structural determinants for activation and block of CFTR-mediated chloride currents by apigenin.

Apigenin (4',5,7-trihydroxyflavone) is an activator of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) currents across epithelia at low concentrations and a blocker at high concentrations. We determined the roles of structural components of apigenin for both stimulation and block of Cl(-) currents across Calu-3 epithelia. The half-maximal binding affinity of apigenin for current stimulation (K(s)) was 9.1 +/- 1.3 microM, and the rank-order of molecular structures was 7-hydroxyl > pyrone = 4'-hydroxyl > 5-hydroxyl. Both the 7-hydroxyl and the 4'-hydroxyl served as H-bond acceptors, whereas the 5-hydroxyl was an H-bond donor. The half-maximal binding affinity of apigenin during current block was 74 +/- 11 microM. Blocked Cl(-) currents were structurally determined by 7-hydroxyl = 4'-hydroxyl > pyrone > 5-hydroxyl. Prestimulation of tissues with forskolin significantly affected activation kinetics and binding characteristics. After forskolin stimulation, K(s) was 4.1 +/- 0.9 microM, which was structurally determined by pyrone > all hydroxyls > single hydroxyls. In contrast, block of Cl(-) current by apigenin was not affected by forskolin stimulation. We conclude that apigenin binds to a stimulatory and an inhibitory binding site, which are distinguished by their affinities and the molecular interactions during binding.

Lactitol-based poly(ether polyol) hydrogels for controlled release chemical and drug delivery systems.

The hydroxyl groups of lactitol were propoxylated to produce poly(ether polyol) (LPEP). The average pK(a) value of hydroxyl groups of the polyol was 1.63. Cross-linked hydrogels were synthesized by esterification with chlorinated poly(ethylene glycol) bis(carboxymethyl) ether (PEGBCOCl). The swelling ratio decreased with increasing cross-linking ratio (PEGBCOCl:LPEP) from 2:1 to 4:1 in the hydrogels and was sensitive to temperature change between 25 and 55 degrees C and concentrations of salt and glucose. The swelling ratio did not change significantly with pH in the range of 4-9. The release profiles of a model active agent, acetylsalicylic acid, from the hydrogels showed that the diffusional release rate had a half-order dependence on time, and the diffusivity decreased with increasing cross-linking ratio. This work demonstrated that LPEP-based hydrogels can be used for controlled delivery of drugs and agrochemicals and the release rates can be controlled with the cross-linking ratio of the hydrogel.

Mechanism and characteristics of protein release from lactitol-based cross-linked hydrogel.

Lactitol-based cross-linked hydrogel was synthesized, and model proteins (alpha-chymotrypsin, beta-lactoglobulin, bovine serum albumin (BSA), and gamma-globulin) were incorporated into the cross-linked hydrogel. The larger-molecular-weight proteins have lower diffusivity (D(e)) in the hydrogel. Increasing temperature accelerated the diffusion rate of proteins; however, the diffusion did not follow the Arrhenius equation at temperatures above 37 degrees C. The swelling ratio of the hydrogel was slightly decreased after heating for 2 h at 37 and 45 degrees C, and significantly reduced after 1 h at 60 degrees C. Therefore, diffusion of beta-lactoglobulin and BSA may be decreased by hydrogel shrinking at temperature over 37 degrees C. The model proteins have high affinities to buffer solution compared to the hydrogel network structure, resulting in high partition coefficients (K > 1) which do not affect the calculation of D(e) values. Incorporated protein release follows the theory of hindered diffusion.

The solid-phase Zincke reaction: preparation of omega-hydroxy pyridinium salts in the search for CFTR activation.

A study of structural modifications of MPB-07 was undertaken as part of a synthetic program aimed at discovering small molecules with CFTR activation potential. Solid-phase synthesis techniques were used to prepare derivatives of MPB-07 employing the Zincke reaction for the construction of aromatic, quaternary ammonium salts such as those found in 2 or 3. In this transformation, primary amines react with highly electrophilic N-2,4-dinitrophenylpyridinium (DNP) salt 4 to afford pyridinium salt 8 with release of 2,4-dinitroaniline 6. Thus, the reaction of 1-(2,4-dinitrophenyl)pyridinium salts with various polymer-bound amino ethers, followed by cleavage from the resin, delivers the desired salts in good yield and high purity.

Diastereoselective synthesis of hydantoin- and isoxazoline-substituted dispirocyclobutanoids.

Synthetic strategies for constructing novel achiral cyclobutanoid isoxazolidinoimidazolidinedione heterocycles, with a generalized structure of II, have been developed via 1,3-dipolar cycloaddition and carbanilide cyclization transformations from methylenecyclobutane 13. The exo methylene cyclobutane system has made the realization of some diasteroselectivity possible, such that the H-bond (Boc-NH) directed product (i.e., 14) was obtained with 3:1 selectivity relative to the non-H-bond directed product (i.e., 15).

Glutathione conjugation of electrophilic metabolites of 1-nitronaphthalene in rat tracheobronchial airways and liver: identification by mass spectrometry and proton nuclear magnetic resonance spectroscopy.

1-Nitronaphthalene (1-NN) is a mutagenic nitroaromatic which has been detected in emissions from both heavy- and light-duty diesel engines, as well as in urban airborne particles. 1-NN is a cytochrome P450-bioactivated, nonciliated bronchiolar epithelial (Clara) cell cytotoxicant. These studies examined the metabolism of 1-NN to electrophilic metabolites which were trapped as glutathione conjugates in highly susceptible (lung) and less susceptible (liver) tissues of the rat. Significant depletion of reduced glutathione was observed at all levels of tracheobronchial airways of rats treated with 200 mg/kg 1-NN, ip. This observation of depleted glutathione was consistent with the HPLC radioactivity profiles demonstrating six glutathione conjugates isolated from liver and lung microsomal incubations with 1-NN, [(3)H]glutathione, and glutathione S-transferase. Mass spectrometry of all six metabolites in electrospray positive ion mode yielded an ion of m/z 497 (M + H), and daughter ions of m/z 479 (loss of water), m/z 306 (glutathione), and m/z 177 (loss of the nitro group and formation of hydroxy naphthalene thiolate ion), demonstrating the formation of hydroxy-dihydroglutathionyl derivatives presumably via intermediate epoxide(s). Proton nuclear magnetic resonance spectroscopy identified four different regioisomeric conjugates from lung and liver microsomal incubations: 1-nitro-7-glutathionyl-8-hydroxy-7, 8-dihydronaphthalene, 1-nitro-7-hydroxy-8-glutathionyl-7, 8-dihydronaphthalene, 1-nitro-5-hydroxy-6-glutathionyl-5, 6-dihydronaphthalene, and 1-nitro-5-glutathionyl-6-hydroxy-5, 6-dihydronaphthalene. HPLC radioactivity profiles demonstrated that major conjugates generated in the lung were derived from the C(7), C(8)-epoxide, whereas the most prominent metabolites in the liver were derived from the C(5),C(6)-epoxide.

Dipolar cycloadditions in solid-phase organic synthesis (SPOS).

We present a concise review of polymer-supported 1,3-dipolar cycloaddition reactions. Nitrile oxide and azomethine ylides constitute the two types of 1,3-dipoles which have been used in conjunction with solid-phase organic synthesis. These cycloaddition reactions on solid phase are generally of equal or greater efficiency than the analogous solution-phase reactions.

Enzyme-linked immunosorbent assay detection of pyrrolizidine alkaloids: immunogens based on quaternary pyrrolizidinium salts.

Polyclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of retrorsine (1, 351 g/mol), monocrotaline (2, 325 g/mol), and retronecine (3, 155 g/mol) in the parts per billion (ppb) range. A set of three bifunctional linking arms (6-8) was synthesized. By N-alkylation of pyrrolizidine alkaloids (PAs) retrorsine, monocrotaline, and retronecine acetonide (9), six haptens (6.1, 6.2, 7.1, 7.2, 7.9, and 8.9) were synthesized and used to generate rabbit antisera. The resulting anti-retrorsine antiserum gave a 50% inhibition (I50) value of 0.9 +/- 0.2 ppb for retrorsine with detection limits of 0.5-10 ppb. The same ELISA system also detected isatidine (4, retrorsine N-oxide) dihydrate (403 g/mol) with an I50 of 1 ppb and senecionine (5,352 g/mol) with an I50 of 100 ppb. A second monocrotaline-based ELISA detected monocrotaline with an I50 of 36 +/- 9 ppb 2 with detection limits of 5-500 ppb and shows no cross-reactivity with 1 or 5; this ELISA demonstrates the potential for the substrate-specific detection method. A third retronecine-based ELISA detects 3 with an I50 of 3000 +/- 600 ppb (3 +/- 0.6 ppm) and detection limits of 600-10,000 ppb. None of these ELISAs cross-react with the structurally similar swainsonine (10) or lupinine (11) alkaloids. PAs were detected in extracts of Senecio vulgaris and Crotalaria retusa, but not in Lupinus spp., as a demonstration of the ELISA's usefulness.

Mass spectrometric analyses of beta-ketolactone oligomers, macrocyclic or catenane structures?

Mass spectrometry is used to develop an analytical method for a new class of organic oligomeric material, beta-ketolactones. Three different series with varying oligomeric sizes are examined. The oligomers may form at least two different structures, a macrocyclic and a catenane ring. Fast atom bombardment coupled with Fourier transform mass spectrometry provides a rapid and convenient method that provides both molecular weight information and abundant fragment ions that are structurally relevant. All the compounds are found to work well with the mass spectrometric analysis. Electrospray ionization coupled with triple-quadrupole mass spectrometry was also evaluated and found to yield results that are similar to FAB. On the basis of the FAB and the low-energy collisionally activated dissociation spectra, we conclude that these compounds are macrocyclic rings and not catenanes.

Mechanism of soluble epoxide hydrolase. Formation of an alpha-hydroxy ester-enzyme intermediate through Asp-333.

18O-Labeled epoxides of trans-1,3-diphenylpropene oxide (tDPPO) and cis-9,10-epoxystearic acid were synthesized and used to determine the regioselectivity of sEH. The nucleophilic nature of sEH catalysis was demonstrated by comparing the enzymatic and nonenzymatic hydrolysis products of tDPPO. The results from single turnover experiments with greater or equal molar equivalents of sEH:substrate were consistent with the existence of a stable intermediate formed by a nucleophilic amino acid attacking the epoxide group. Tryptic digestion of sEH previously subjected to multiple turnovers with tDPPO in H2 18O resulted in the isolation and purification of a tryptic fragment containing Asp-333. Electrospray mass spectrometry of this fragment conclusively illustrated the incorporation of 180. After complete digestion of the latter peptide it was shown that Asp-333 of sEH exhibited an increased mass. The attack by Asp-333 initiates enzymatic activity, leading to the formation of an alpha-hydroxyacyl-enzyme intermediate. Hydrolysis of the acyl-enzyme occurs by the addition of an activated water to the carbonyl carbon of the ester bond, after which the resultant tetrahedral intermediate collapses, yielding the active enzyme and the diol product.

Improved radiolabeled substrates for soluble epoxide hydrolase.

Two rapid assays for the soluble epoxide hydrolase (sEH) are described. First, a sensitive radiometric assay based on thin-layer chromatography of [(14)C]-cis-9,10-epoxystearic acid and its corresponding diol ((14)C]-9,10-dihydroxystearic acid) is described. The cis fatty acid oxide exhibits higher specific activity of hydration with sEH from mouse, rat, human, and potato compared to trans-stilbene oxide (TSO). The K(m) and V(max) obtained for [(14)C]-cis-9,10-epoxystearic acid with mouse sEH are 11.0 microM and 3460 nmol/min/mg protein, respectively. [(14)C]-cis-9,10- Epoxystearic acid might more closely mimic the structures of natural substrates for sEH. Second, [2-(3)H]-trans-1,3-diphenyl-propene oxide ([(3)H]-tDPPO) and [2-(3)H]-cis-1,3-diphenylpropene oxide ([(3)H]-cDPPO) were synthesized and rapid radiometric assays for epoxide hydrolases (EHs) were developed by differential partitioning of the epoxide into iso-octane and its corresponding diol into aqueous phase containing methanol. It was shown that sEHs from mouse, rat, human, and potato rapidly hydrolyze [(3)H]-tDPPO and in comparison to TSO have 20-,49-,28-, and 7-fold higher rates, respectively. Mouse sEH hydrates [(3)H]-tDPPO at 26,200 nmol/min/mg protein, and a K(m)p4 of 2.80 microM is observed.

Development of surrogate substrates for neuropathy target esterase.

Seventeen substrates were synthesized and their activities as surrogate substrates for Neuropathy Target Esterase were tested. Substrates investigated are carbon analogs of phenylvalerate (1) with oxygen and sulfur substituted at the alpha, beta and gamma positions. Phenol and thiophenol esters of these analogs constitute two series of compounds tested. The ratio of catalytic hydrolysis to background hydrolysis increased at lower pH values with all substrates tested including phenylvalerate (1). There was more than a 2.5-fold increase in specific activity with phenylthiopropylethanoate (6) at pH of 6.75 compared to phenylvalerate (1). Furthermore, a 19-fold decrease in Km is reported with compound 6. This and related compounds can be used as the basis of more sensitive assays for neuropathy target esterase. Thiophenyl esters in this series are sufficiently good substrates to hold promise in continuous assays.