Catheter ablation in atrial fibrillation: is there a mortality benefit in patients with diabetes and heart failure?

Atrial fibrillation (AF) is the most commonly sustained arrhythmia, and patients with diabetes mellitus (DM) exhibit an increased incidence of AF. Besides DM, heart failure (HF) shares pathophysiological links with AF, mainly related to the pathological remodeling of hearts affected by structural disease. As in a vicious circle, AF may contribute to HF worsening and increased mortality in patients with structural heart diseases, and the outcome may be further impaired when concomitant DM is present. Although no data directly referring to DM patients with HF are available, indirect information can be drawn from large studies on patients with HF and AF. The present review discusses the outcome of AF ablation in patients with DM and HF, focusing on safety, efficacy, and most particularly on hard endpoints such as mortality and thromboembolic event incidence.

Structure and heterogeneity of the one- and two-disulfide folding intermediates of tick anticoagulant peptide.

Tick anticoagulant peptide (TAP) is a factor Xa-specific inhibitor and is structurally homologous to bovine pancreatic trypsin inhibitor (BPTI). The fully reduced TAP refolds spontaneously to form the native structure under a wide variation of redox buffers. The folding intermediates of TAP consist of at least 22 fractions of one-disulfide, two-disulfide, and three-disulfide scrambled isomers. Three species of well-populated one- and two-disulfide intermediates were isolated and structurally characterized. The predominant one-disulfide species contains TAP-(Cys33-Cys55). Two major two-disulfide isomers were TAP-(Cys33-Cys55, Cys15-Cys39) and TAP-(Cys33-Cys55, Cys5-Cys39). Both Cys33-Cys55 and Cys15-Cys39 are native disulfides of TAP. These three species are structural counterparts of BPTI-(Cys30-Cys51), BPTI-(Cys30-Cys51, Cys14-Cys38), and BPTI-(Cys30-Cys51,Cys5-Cys38), which have been shown to be the major intermediates of BPTI folding. In addition, time-course-trapped folding intermediates of TAP, consisting of about 47% one-disulfide species and 30% two-disulfide species, were collectively digested with thermolysin, and fragmented peptides were analyzed by Edman sequencing and mass spectrometry in order to characterize the disulfide-containing peptides. Among the 15 possible single-disulfide pairings of TAP, 10 (2 native and 8 nonnative) were found as structural components of its one- and two-disulfide folding intermediates. The results demonstrate that the major folding intermediates of TAP bear structural homology to those of BPTI. However, the folding pathway of TAP differs from that of BPTI by (a) a higher degree of heterogeneity of one- and two-disulfide intermediates and (b) the presence of three-disulfide scrambled isomers as folding intermediates. Mechanism(s) that may account for these diversities are proposed and discussed.

The structure of denatured bovine pancreatic trypsin inhibitor (BPTI).

In the presence of denaturant and thiol initiator, the native bovine pancreatic trypsin inhibitor (BPTI) denatures by shuffling its native disulfide bonds and converts to a mixture of scrambled isomers. The extent of denaturation is evaluated by the relative yields of the scrambled and native species of BPTI. BPTI is an exceedingly stable molecule and can be effectively denatured only by guanidine thiocyanate (GdmSCN) at concentrations higher than 3-4 M. The denatured BPTI consists of at least eight fractions of scrambled isomers. Their composition varies under increasing concentrations of GdmSCN. In the presence of 6 M GdmSCN, the most predominant fraction of scrambled BPTI accounts for 56% of the total structure of denatured BPTI. Structural analysis reveals that this predominant fraction contains the bead-form isomer of scrambled BPTI, bridged by three pairs of neighboring cysteines, Cys5-Cys14, Cys30-Cys38 and Cys51-Cys55. The extreme conformational stability of BPTI has important implications in its distinctive folding pathway.

Direct analysis of drugs by continuous-flow fast-atom bombardment and tandem mass spectrometry.

The techniques of continuous-flow fast-atom bombardment (CF-FAB) and tandem mass spectrometry (MS/MS) are combined and applied to the analysis of small molecular mass drugs (mol.wt less than 500 Da). The approach involves the interfacing of a CF-FAB inlet with a triple-stage quadrupole mass spectrometer, enabling the acquisition of collision-activated decomposition mass spectra of the drugs after FAB ionization. The relationship between a stable sample surface on the CF-FAB probe tip and the quality of the mass spectrum is discussed, as are practical methods for obtaining and maintaining surface stability. CF-FAB MS/MS spectra for several drugs are presented, including penicillin G, phentolamine, cocaine and benzoylecgonine. Minimum detection limits range from 50-500 pg injected, depending on the compound. The reproducibility of the integrated areas of peaks from repetitive injections is approximately five per cent. Data are also presented for the direct CF-FAB MS/MS analysis of cocaine and benzoylecgonine in spiked urine samples.

Carcinogenicity and metabolic activation of hexestrol.

The carcinogenic activity of the synthetic estrogen hexestrol was measured in male Syrian hamsters. Between 90% and 100% of the animals treated with hexestrol or with 3',3",5',5"-tetradeuteriohexestrol, implanted subcutaneously as 25-mg pellets, were found with renal carcinoma after 6-7 months. In vitro hexestrol metabolism, mediated by phenobarbital-induced rat liver microsomes, led to the formation of 3'-hydroxyhexestrol. This metabolite was identified by comparison with authentic reference material synthesized by oxidation of hexestrol with Fremy's salt. Diethylstilbestrol could not be detected as a metabolite. In urine of male Syrian hamsters, 3'-hydroxyhexestrol, 3'-methoxyhexestrol, 1-hydroxyhexestrol, and other hydroxylated and/or methoxylated hexestrol metabolites were identified. Again, diethylstilbestrol was not detectable as a hexestrol metabolite in vivo. The reactivity of 3'-hydroxyhexestrol was then studied to determine if this catechol estrogen played a role in hexestrol carcinogenicity. Horseradish peroxidase catalyzed the oxidation of 3'-hydroxyhexestrol to 3',4'-hexestrol quinone. This oxidation reaction could also be carried out non-enzymatically using silver oxide or silver carbonate on celite as oxidants. The quinone was unstable (t1/2 in methylene chloride: 53 min). It reacted with sulfur-containing compounds such as mercaptoethanol by Michael addition to form 3'-(2-hydroxyethylthio)-5'-hydroxyhexestrol. 3',4'-Hexestrol quinone reacted with simple amines such as ethylamine to form N-ethyl-aminohexestrol. The chemical reactions described above were carried out to test the reactivity of identified or suspected metabolic intermediates of hexestrol. It was concluded that carcinogenicity of hexestrol was not based on its conversion to diethylstilbestrol. Rather, catechol estrogen formation may be necessary for the carcinogenic action of hexestrol in analogy to events observed earlier with estradiol.

Reactivity of 4',4"-diethylstilbestrol quinone, a metabolic intermediate of diethylstilbestrol.

In a search for the carcinogenic metabolite of diethylstilbestrol, the interactions of 4',4"-diethylstilbestrol quinone with peptides and nucleic acids were investigated. Nonextractable binding of 4',4"-diethylstilbestrol quinone to calf thymus DNA or poly G were observed. However, adduct nucleosides could not be isolated subsequent to enzymatic digestion of nucleic acids. Binding to dGMP or dAMP also occurred, but the initially bound stilbene estrogen could mostly be extracted with 18 extractions using various organic solvents. Non-covalent interactions of 4',4"-diethylstilbestrol quinone with calf thymus DNA were observed spectrally only after exhaustive dialysis of the DNA versus water, but not with native DNA. In chemical reactions of 4',4"-diethylstilbestrol quinone and nucleosides, nucleotides, and amines such as n-pentyl amine, only Z,Z-dienestrol could be identified as reaction product. The quinone did react with mercaptoethanol via Michael addition to the unsaturated carbonyl system to form a stable adduct, 4-(2-hydroxyethylthio)-3,4-di(p-hydroxyphenyl)-2-hexene. It also reacted covalently with sulfur-containing peptides such as reduced glutathione or bovine serum albumin. Partially purified rat liver cytochrome P-450 reductase reduced 4',4"-diethylstilbestrol quinone to E- and Z-diethylstilbestrol. It is proposed that 4',4"-diethylstilbestrol quinone forms unstable adduct intermediates with DNA which decompose with time. Also, covalent binding of 4',4"-diethylstilbestrol quinone to important proteins via thioether linkages may play a role in carcinogenesis.

Diethylstilbestrol (DES) quinone: a reactive intermediate in DES metabolism.

The quinone of E-diethylstilbestrol (DES), a postulated metabolic intermediate derived from DES, has been synthesized by oxidation of DES in chloroform using silver oxide. The reaction product was structurally characterized by infrared, ultraviolet, nuclear magnetic resonance, and mass spectrometry. The product of oxidation of DES by hydrogen peroxide, catalyzed by horseradish peroxidase and also by rat uterine peroxidase, was shown to be identical with synthetic DES quinone based on identical u.v. spectra and on identical decomposition products. DES quinone was stable only in non-protic solvents such as chloroform. In acids, bases or protic solvents, DES quinone rearranged to Z,Z-dienestrol (beta-DIES). The half-life of DES quinone in water was approximately 40 min; in methanol it was approximately 70 min. Bacterial mutagenicity (Ames) tests did not indicate that DES quinone had mutagenic or genotoxic activity. However, DES quinone was found to bind to calf thymus DNA without any enzyme mediation at levels significantly above the binding of DES under the same conditions. Based on the binding of DES quinone to DNA, this intermediate must be considered as a possible carcinogenic metabolite of DES.

Mechanism of diethylstilbestrol carcinogenicity as studied with the fluorinated analogue E-3',3",5',5"-tetrafluorodiethylstilbestrol.

E-3',3",5',5"-Tetrafluorodiethylstilbestrol (TF-DES), a structural analogue of diethylstilbestrol synthesized as a possible noncarcinogenic estrogen, was found to induce renal clear-cell carcinoma in Syrian hamster. The tumor induction frequency of TF-DES was the same as that of diethylstilbestrol, although the induction period was longer (approximately 9 months) than that of diethylstilbestrol (approximately 6 months). TF-DES was estrogenic and was found to support in vivo growth of estrogen-dependent H-301 cells, a cell line derived from the primary estrogen-induced and -dependent renal clear-cell carcinoma of male Syrian hamster. These data established the ability of TF-DES not only to induce tumors but also to promote estrogen-dependent tumor growth after the initiation process. Oxidation of TF-DES to TF-DES quinone was catalyzed by horseradish peroxidase. The structure of this metabolic intermediate was confirmed by comparison with synthesized TF-DES quinone. This intermediate was very unstable (half-life in methanol, 24 min; half-life in water, 4 min) and rearranged to Z,Z-3',3",5',5"-tetrafluorodienestrol. Based on the experiments with the fluorinated derivative, it is postulated that stilbestrol estrogens induce tumors via metabolic oxidation to quinone intermediates, which then may interact with DNA or other cellular targets.

Synthesis and spectroscopic analysis of modified bile salts.

For the study of hepatic bile acid transport in vivo, a series of modified bile salts were synthesized. The N-cholyl derivatives of L-leucine, L-alanine, D-alanine, beta-alanine, L-proline, and gamma-amino-butyric acid were prepared from cholic acid, ethyl chloroformate and the corresponding amino acid. Structural analysis of products was carried out mainly by electron impact mass spectrometry (20 eV) of the methyl ester/acetate derivatives. In all EI spectra, fragments in the lower mass region included McLafferty rearrangement ions (beta-cleavage) and product ions of gamma-cleavage in the vicinity of the amide linkage. In the upper mass region, fragmentation was characterized by consecutive eliminations of ketene and/or acetic acid from low intensity molecular ions. The purity of the products and their molecular weights were checked by a novel ionization technique in mass spectrometry, fast atom bombardment (FAB) mass spectrometry. FAB spectra were obtained from underivatized bile salts. The spectra were characterized by ions formed by attachment of a proton or an alkali ion to the bile salt to give intense M+H, M+Na, or M+K ions, which then showed little fragmentation.

Deuterium labeling of diethylstilbestrol and analogues.

E-2,2,3',3'',5,5,5',5''-octadeuteriodiethylstilbestrol (DES-d8) and Z-2,3'3'',4,5,5,5',5''-octadeuterio-3,4-bis(p-hydroxyphenyl)-2-hexene (psi-DES-d8) were synthesized from E-diethylstilbestrol (DES) by hydrogen/deuterium exchange in a mixture of methanol-d and deuterium chloride in deuterium oxide. The structures, isotopic purity, and positions of uptake of deuterium were determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS). Additional confirmation of the positions of deuterium exchange in stilbestrols was obtained from an analysis of the oxidation of dES-d8 to Z,Z-2,3',3'',5,5',5''-hexadeuteriodienestrol (beta-DIES-d6) and of the hydrogen/deuterium exchange reaction of hexestrol (HEX) to 3',3'',5',5''-hexestrol, (HEX-d4). Structural analysis and the determination of isotopic purity of the latter two compounds were also carried out by NMR and MS. The uptake of eight deuterium atoms by DES is postulated to proceed via two different reactions occurring simultaneously: 1. acid catalyzed deuteration of all four phenolic ortho-positions (3',3'',5',5''); 2. acid catalyzed deuteration of the olefin bridge with subsequent formation of deuterated psi-DES (3 or 4). Due to the equilibration between DES, psi-DES, and Z-diethylstilbestrol (cis-DES) in the acidic reaction mixture at 85 degrees C, the deuterated psi-DES is thought to rapidly rearrange to deuterated DES. Repeated deuteration will eventually form DES-d8 fully labeled in the 2,2,5,5 methylene positions.

Fast atom bombardment mass spectrometry of estrogen glucuronides and sulfates.

Fast atom bombardment (FAB) mass spectra of 13 intact, underivatized glucuronides and/or sulfate salts are reported. Spectra are characterized by abundant ions formed by attachment of a proton, [M+H]+, or of an alkali ion, [M+alkali]+, to the glucuronide or sulfate salt. Fragment ions were of low intensity. FAB spectra can be used to obtain the molecular weight of a sample, to assess its purity and to identify the nature of the alkali of the glucuronide or sulfate salt.