Maximal enzyme activities, and myoglobin and glutathione concentrations in heart, liver and skeletal muscle of the Northern Short-tailed shrew (Blarina brevicauda; Insectivora: Soricidae).

We measured the enzymes of glycolysis, Krebs Cycle, beta-oxidation and electron transport in the heart, liver and skeletal muscle of the Northern Short-tailed Shrew, Blarina brevicauda. Additionally, we measured the amount of myoglobin in skeletal and heart muscle as well as the concentration of glutathione in heart. The picture that emerges is of an aerobically well-endowed animal with constrained anaerobic capacity as indicated by small activities of glycolytic enzymes and creatine kinase. Lipid metabolism and amino acid transamination, as well as gluconeogenesis, are predominant in processing carbon resources and probably reflect the large contribution lipid and protein make to the diet of this carnivore. The citrate synthase activity is the largest of any reported value for vertebrate heart (250 U/g). The additional, very active cytochrome c oxidase activity (220 U/g) and large myoglobin concentrations (8 mg/g) in heart are clearly the underpinnings of the rapid metabolic rates reported for small insectivores. The potential for generation of reactive oxygen species must be great since the total glutathione concentration (165 mumol/g) is 300-fold greater in shrew hearts than in hearts of rats.

Unusually weak oxygen binding, physical properties, partial sequence, autoxidation rate and a potential phosphorylation site of beluga whale (Delphinapterus leucas) myoglobin.

We purified myoglobin from beluga whale (Delphinapterus leucas) muscle (longissimus dorsi) with size exclusion and cation exchange chromatographies. The molecular mass was determined by mass spectrometry (17,081 Da) and the isoelectric pH (9.4) by capillary isoelectric focusing. The near-complete amino acid sequence was determined and a phylogeny indicated that beluga was in the same clad as Dall's and harbor porpoises. There were consensus motifs for a phosphorylation site on the protein surface with the most likely site at serine-117. This motif was common to all cetacean myoglobins examined. Two oxygen-binding studies at 37 degrees C indicated dissociation constants (20.5 and 23.6 microM) 5.7-6.6 times larger than horse myoglobin (3.6 microM). The autoxidation rate of beluga myoglobin at 37 degrees C, pH 7.2 was 0.218+/-0.028 h(-1), 1/3 larger than reported for myoglobin of terrestrial mammals. There was no clear sequence change to explain the difference in oxygen binding or autoxidation although substitutions (N66 and T67) in an invariant rich sequence (HGNTV) distal to the heme may play a role. Structural models based on the protein sequence and constructed on topologies of known templates (horse and sperm whale crystal structures) were not adequate to assess perturbation of the heme pocket.

The heart and estrogen/progestin replacement study revisited: hormone replacement therapy produced net harm, consistent with the observational data.

Lower coronary event rates in women receiving hormone replacement therapy (HRT) have led to a presumption of benefit. The Heart and Estrogen/Progestin Replacement Study, a large randomized trial, observed a 1.4% first year excess of coronary events, well beyond the plausible play of chance on the expected effect. Over the duration of the study, event totals were similar, but patients treated with HRT experienced them earlier, with a net loss of patient-months of event-free survival. The point at which the lower event rate in hormone-treated patients would fully repay the first year loss, with constant rates, is almost double the trial duration (of 4.1 years). Since patients in the trial were preselected for satisfactory adherence to therapy, the net benefit in practice is likely to be even less. Had the patients in the Heart and Estrogen/Progestin Replacement Study been recruited to an observational study at various intervals over the first 5 years after starting HRT, the apparent risk reduction over 5 years would have been between 21% and 34%. A previous meta-analysis of trials of HRT for other indications also shows net harm. Women with or at high risk of coronary heart disease should not start HRT. There is a risk that women without coronary heart disease might experience even greater net harm from HRT. The late benefit is necessarily limited, as it cannot exceed the event rate. The mechanism of the early loss is unknown; if it were reduced proportionately less than the late benefit, considerable net harm could result. Arch Intern Med. 2000;160:2897-2900

Long chain fatty acids inhibit and medium chain fatty acids activate mammalian cardiac hexokinase.

We investigated the effect of non-esterified fatty acids (FAs) on bovine heart hexokinase (type I: ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1). Long chain FAs (C14 to C20) inhibited the enzyme in a way that correlated positively with both the chain length and the degree of unsaturation. Medium chain FA with 12 or less carbons activated hexokinase in a chain length dependent manner with the greater activation shown by laurate. The activation constant of laurate was 91.5 microM with a maximal activation of 60.3%. Oleate caused a maximal decrease in specific activity of 25% with an inhibition constant of 79 microM. Using the fluorescent probe cis-parinarate, we found a saturable binding site with K(d) of 3.5 microM. Oleate competed the fluorescent probe from the protein with a K(d) of 1.4 microM. Medium chain FAs did not compete the probe from HK. The binding of fatty acid to the protein appears to be entropically driven as indicated by an Arrhenius analysis (DeltaS=+231.6 J mol(-1) deg(-1)). The presence of oleate significantly increased the K(ATP)(m) from 0.47 mM to 0.89 mM while the K(glucose)(m) in the presence of the FA (0.026+/-0.003 mM) was not significantly different from the control (0.014+/-0.004 mM). A decrease in V(max) values in the presence of oleate indicated that a mixed allosteric inhibition was operating.

The interaction of ferrocytochrome c with long-chain fatty acids and their CoA and carnitine esters.

Non-covalent modification of cytochrome c may have implications for electron transport and energy metabolism. We examined the interaction of various fatty acids (FAs), their coenzyme A and carnitine esters, and fatty alcohols with horse heart ferrocytochrome c. A comparison of FAs indicated a minimum chain length of 14 carbons was required for significant effect on the ferroheme chromophore and major changes in electronic spectra. Coenzyme A and carnitine esters interacted less strongly than FAs whereas long-chain alcohols did not interact with the protein. We found a single, saturable FA binding site with Kd (oleate) of 23.1 microM (by stopped-flow kinetics), 30 microM (by radiochemical binding assay), and 29 microM (by spectrophotometric assay). The binding stoichiometry was 1:1. We present evidence from electronic spectra, and proton NMR (nuclear magnetic resonance) that the S-Fe coordination (methionine 80) was disrupted by ligand binding. From molecular modeling we identify a putative binding channel flanked by lysines 72 and 73.

Anticoagulation in chronic nonvalvular atrial fibrillation: appraisal of two meta-analyses.

Five randomized trials of warfarin stroke prophylaxis in atrial fibrillation have undergone meta-analyses by the Atrial Fibrillation Investigators (AFI) and by the British Columbia Office of Health Technology Assessment (BCOHTA), with differing conclusions. The AFI, using the original data, applied a consistent definition of 'major' bleeding (intracranial, hospitalization or transfusion of at least 2 U of blood) and found an excess of six major bleeding events. The BCOHTA used the definitions used in the studies, including "any medical intervention", and counted an excess of 21 'major' bleeding events. They then compared these with only the most severe one-third of the strokes. The BCOHTA were concerned that lack of blinding may have influenced the diagnosis of mild stroke, but the data do not suggest diagnostic bias. The risk reduction in the BCOHTA analysis of the most severe one-third of strokes was almost identical to that in the remaining strokes. The value of treatment is best assessed by comparing good with bad events of similar impact, and eliminating strokes from analysis does not eliminate them from patients. The BCOHTA analysis confirms the risk reduction demonstrated by the AFI.

The accuracy of predicting Thoroughbred heart scores.

Veterinarians commonly predict mature heart scores for yearling and 2-year-old horses to aid clients in assessing a horse's racing potential. Sixty-six thoroughbreds were assessed as a yearlings or 2-year-olds, then re-assessed as a mature horse (over the age of 3). Of these horses, 82% of the yearlings and 87% of the 2-year-olds had a correctly predicted mature heart score within one heart score range (i.e. three points).

The Canadian American Ticlopidine Study (CATS) in thromboembolic stroke.

The Canadian American Ticlopidine Study (CATS) is a randomised, double-blind, placebo-controlled trial to assess the effect of ticlopidine (250 mg twice daily) in reducing the rate of subsequent occurrence of stroke, myocardial infarction, or vascular death in patients who have had a recent thromboembolic stroke. Twenty-five centres entered 1072 patients into the study between 1 week and 4 months after their qualifying stroke. The patients were treated and followed for up to 3 years (mean 24 months). In the efficacy analysis, the event rate per year for stroke, myocardial infarction or vascular death, considered together, was 15.3% in the placebo group and 10.8% in the ticlopidine group, representing a relative risk reduction with ticlopidine of 30.2% (95% confidence interval 7.5-48.3%; p = 0.006). Ticlopidine was beneficial for both men and women (relative risk reductions 28.1%, p = 0.037, and 34.2%, p = 0.045, respectively). Analysis by intention-to-treat gave a smaller estimate of risk reduction (23.3%, p = 0.020) for stroke, myocardial infarction, or vascular death. Adverse experiences associated with ticlopidine included neutropenia (severe in about 1% of cases) and skin rash and diarrhoea (severe in 2% of cases each); all were reversible. This study provides evidence of a beneficial effect of ticlopidine in both men and women with a recent thromboembolic stroke.

The Canadian American Ticlopidine Study (CATS) in thromboembolic stroke. Design, organization, and baseline results.

The Canadian American Ticlopidine Study is a randomized, placebo-controlled, double-blind, multicenter study to assess the efficacy and safety of ticlopidine hydrochloride in patients who have suffered a thromboembolic stroke no less than 1 week and no more than 4 months before entry into the study. The primary assessment of efficacy will be based on the cluster of outcome events recurrent stroke, myocardial infarction, or vascular death. Twenty-five clinical centers, 12 in Canada and 13 in the United States, entered a total of 1,072 patients during a 3-year recruitment period; these patients were randomly allocated to receive either 250 mg ticlopidine or identical-appearing placebo tablets twice daily for up to 3 years. Patient recruitment was completed in December 1986. Patients were followed for a maximum of 3 years or until the close of the study in December 1987; at that time an average follow-up of 25 months had been achieved. We summarize the protocol and organization of the study and document the methods of execution and analysis, with corresponding criteria, before disclosure of the treatment code to any of the study investigators. We also provide a clinical description of the patients at entry into the study.

A secondary prevention, randomized trial of suloctidil in patients with a recent history of thromboembolic stroke.

Four hundred and thirty-eight patients who had suffered a thromboembolic stroke not less than two weeks or more than four months previously, were entered into a placebo-controlled randomized clinical trial to determine whether suloctidil (200 mg t.i.d.) would influence the subsequent recurrence of stroke, the occurrence of myocardial infarction, or cardiovascular death. The two treatment groups were comparable at baseline with respect to important prognostic variables and there was good adherence to the study protocol during an average follow-up of 20 months. Significantly more patients complained of side-effects in the suloctidil group and more hepatotoxicity was also reported in the suloctidil group. Four cases of clinical hepatitis were suspected to be due to suloctidil, each of which was reversible on termination of study treatment; relative increases in SGOT and SGPT at three months in the suloctidil group were found to be mild and transient. The primary analysis of efficacy was based on the incidence of the first event of stroke, myocardial infarction or cardiovascular death, but excluding events that occurred more than 28 days after complete withdrawal from study medication for whatever reason. Thus, the primary analysis included 38 events in the suloctidil group and 47 in the placebo group (p = 0.17) representing a risk reduction of 24%. If total mortality is substituted for cardiovascular death, the corresponding figures are 47 in the suloctidil group and 58 in the placebo group (p = 0.08).(ABSTRACT TRUNCATED AT 250 WORDS)