8-epi PGF2 alpha generation during coronary reperfusion. A potential quantitative marker of oxidant stress in vivo

BACKGROUND: Myocardial reperfusion is believed to be associated with free radical injury. However, indexes of oxidative stress in vivo have been limited by their poor specificity and sensitivity. Isoprostanes are stable products of arachidonic acid formed in a nonenzymatic, free radical-catalyzed manner. We have developed a sensitive and specific assay for one of these compounds, 8-epi prostaglandin (PG) F2 alpha. METHODS AND RESULTS: To address its utility as an index of oxidative stress during coronary reperfusion, we measured urinary levels by gas chromatography/mass spectrometry in a canine model of coronary thrombolysis, in patients with acute myocardial infarction treated with thrombolytic therapy, and in patients after elective coronary artery bypass surgery. Urinary 8-epi PGF2 alpha was unchanged after circumflex artery occlusion in a canine model of coronary thrombolysis (n = 13; 437.2 +/- 56.4 versus 432.7 +/- 55.2 pmol/mmol creatinine) but increased significantly (P < .05) immediately after reperfusion (553.8 +/- 64.7 pmol/mmol). Urinary levels were increased (P < .001) in patients (n = 12) with acute myocardial infarction given lytic therapy (265.8 +/- 40.8 pmol/mmol) compared with age-matched control subjects (n = 20; 91.5 +/- 11.8 pmol/mmol) and patients with stable coronary disease (n = 20; 95.7 +/- 6.3 pmol/mmol). Preoperative levels rose from 113.2 +/- 11.8 to 248.2 +/- 86.3 pmol/mmol at 30 minutes into revascularization to 332.2 +/- 82.6 pmol/mmol by 15 minutes after global myocardial reperfusion (P < .05) and dropped to 181.2 +/- 50.4 pmol/mmol at 30 minutes and 120.2 +/- 9.9 pmol/mmol at 24 hours after bypass surgery (n = 5). Corresponding changes in spin adduct formation, found with electron paramagnetic resonance, were noted in 2 patients. CONCLUSIONS: These data support the hypothesis that free radical generation occurs during myocardial reperfusion. Measurement of isoprostane production may serve as a noninvasive index of oxidative stress.     

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as a substrate of cytochrome P450 2D6: allosteric effects of NADPH-cytochrome P450 reductase

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that produces Parkinsonism symptoms in man, has been examined as a substrate of recombinant human cytochrome P450 2D6. When cumene hydroperoxide is used as an oxygen and electron donor, a single product is formed, identified as 4-phenyl-1,2,3,6-tetrahydropyridine. The K(m) for formation of this product (130 microM) is in agreement with the dissociation constants for MPTP binding to the enzyme determined by optical and nuclear magnetic resonance (NMR) spectroscopy. When the reaction is carried out with nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and recombinant human NADPH-cytochrome P450 reductase, a second product, identified as 1-methyl-4-(4'-hydroxyphenyl)-1,2,3,6-tetrahydropyridine, is formed in addition to 4-phenyl-1,2,3,6-tetrahydropyridine. The K(m) values for formation of these two products are 19 microM and 120 microM, respectively. Paramagnetic relaxation experiments have been used to measure distances between the protons of bound MPTP and the heme iron, and these have been used to construct models for the position and orientation of MPTP in the active site. For the cytochrome alone, a single mode of binding was observed, with the N-methyl close to the heme iron in a position appropriate for the observed N-demethylation reaction. In the presence of the reductase, the data were not consistent with a single mode of binding but could be explained by the existence of two alternative orientations of MPTP in the active site. One of these, characterized by a dissociation constant of 150 microM, is essentially identical to that observed in the absence of the reductase. In the second, which has a K(d) of 25 microM, the MPTP is oriented so that the aromatic ring is close to the heme iron, in a position appropriate for p-hydroxylation leading to the formation of the product seen only in the presence of the reductase. In the case of codeine, another substrate for cytochrome P450 2D6, the addition of reductase had no effect on the nature of the product formed, the dissociation constant, or the orientation in the binding site. These observations show that NADPH-cytochrome P450 reductase has an allosteric effect on the active site of cytochrome P450 2D6 that affects the binding of some substrates but not others.     

Stable Oxygen and Carbon Isotopic Compositions of Lates stappersii Otoliths from Lake Tanganyika,East Africa

In this study, we measured growth trends in oxygen and carbon isotopic ratios in whole sagittal otoliths from three adult centropomid fish (Lates stappersii) from each of three sub-basins of Lake Tanganyika, East Africa. Sampling density was 20 to 50 samples per otolith. Both δ¹⁸O and δ¹³C values increase with age. The δ¹⁸O data suggest that otoliths were precipitated near the expected equilibrium with the ambient environment (ca. +3.5‰) and support a migration pattern from surface waters during larval stages to deeper waters (40 to 80 m) for mature fish. Relatively high δ¹⁸O values in the southern sub-basin are consistent with cooler temperatures in the region during seasonal upwelling. The δ13C increase from otolith core to edge is large (up to 4‰) and is interpreted as due to ontogenetic changes in diet and contributions from a decrease in the proportion of respired CO₂ incorporated into otolith carbonate as metabolic rates of the fish dropped with maturity. The data seem to successfully reveal life strategy and migration patterns of L. stappersii, document regional differences in lake conditions, and provide a record of temperature within the water column during which the fish lived. Higher resolution studies and analyses of historical samples could be used to constrain modern and past growth patterns, and to reconstruct past temperature gradients and productivity patterns in the lake.     

Efficient design of oversampled NPR GDFT filterbanks

We propose a flexible, efficient design technique for the prototype filter of an oversampled near perfect reconstruction (NPR) generalized discrete Fourier transform (GDFT) filterbank. Such filterbanks have several desirable properties for subband processing systems that are sensitive to aliasing, such as subband adaptive filters. The design criteria for the prototype filter are explicit bounds (derived herein) on the aliased components in the subbands and the output, the distortion induced by the filterbank, and the imaged subband errors in the output. It is shown that the design of an optimal prototype filter can be transformed into a convex optimization problem, which can be efficiently solved. The proposed design technique provides an efficient and effective tool for exploring many of the inherent tradeoffs in the design of the prototype filter, including the tradeoff between aliasing in the subbands and the distortion induced by the filterbank. We calculate several examples of these tradeoffs and demonstrate that the proposed method can generate filters with significantly better performance than filters obtained using current design methods.     

Reprocessing of Microcrystalline Cellulose Spheres with Low Drug Concentrations

Spherical dosage forms have been reported to be an efficient and effective method for delivering drugs into the body and controlling their dissolution rate. Substantial work has been conducted in these laboratories illustrating the advantages of microcrystalline cellulose-based spheres for these purposes. Through various methodologies, but most routinely the extrusion and marumerization technique, it has been determined by Funck, et al. that not more than 50% drug can be incorporated into a sphere formulation without the addition of other binders.

Because of the nature of the extrusion and marumerization manufacturing process, the type of drug being processed and the resultant particle size requirements of the spheres, the percent of spheres falling outside the desired particle size can range from between 3% and 20%. For this reason and the possibility of operator errors, our objective was to determine the parameters which needed monitoring when reprocessing was necessary.