Mutations in beta-spectrin disrupt axon outgrowth and sarcomere structure.

beta-Spectrin is a major component of the membrane skeleton, a structure found at the plasma membrane of most animal cells. beta-Spectrin and the membrane skeleton have been proposed to stabilize cell membranes, generate cell polarity, or localize specific membrane proteins. We demonstrate that the Caenorhabditis elegans homologue of beta-spectrin is encoded by the unc-70 gene. unc-70 null mutants develop slowly, and the adults are paralyzed and dumpy. However, the membrane integrity is not impaired in unc-70 animals, nor is cell polarity affected. Thus, beta-spectrin is not essential for general membrane integrity or for cell polarity. However, beta-spectrin is required for a subset of processes at cell membranes. In neurons, the loss of beta-spectrin leads to abnormal axon outgrowth. In muscles, a loss of beta-spectrin leads to disorganization of the myofilament lattice, discontinuities in the dense bodies, and a reduction or loss of the sarcoplasmic reticulum. These defects are consistent with beta-spectrin function in anchoring proteins at cell membranes.

Experimental and field sulfaquinoxaline toxicosis in Leghorn chickens.

Mortality of 47% occurred in a commercial flock of 20-week-old leghorn pullets treated with 0.05% sulfaquinoxaline in the feed. The drug was given for 2-3 days, removed for 2 days, and fed again for 2-3 days. Gross lesions consisted of disseminated hemorrhages, bone marrow pallor, a variety of changes suggesting septicemia, and overwhelming bacterial infection. Experimental feeding of sulfaquinoxaline at 0.05% in the feed at a schedule of 3 days on, 3 days off, and 2 days on successfully reproduced the essential features and gross lesions of the field toxicosis. Mortalities of 32.5% and 43.5% were observed in sulfaquinoxaline-treated groups. A high incidence of gangrenous dermatitis occurred in chickens with the experimental toxicosis, but it was not observed in the field case.

Non-linearities in the pharmacokinetics of di-(2-ethylhexyl) phthalate and metabolites in male rats.

The disposition of the plasticizer di-(2-ethylhexyl) phthalate (DEHP) and four of its major metabolites was studied in male rats given single infusions of a DEHP emulsion in doses of 5, 50 or 500 mg DEHP/kg body weight. Plasma concentrations of DEHP and metabolites were followed for 24 h after the start of the infusion. The kinetics of the primary metabolite mono-(2-ethylhexyl) phthalate (MEHP) was studied separately. The concentrations of DEHP in plasma were at all times considerably higher than those of MEHP, and the concentrations of MEHP were much higher than those of the other investigated metabolites. In animals given 500 mg DEHP/kg, the areas under the plasma concentration-time curves (AUCs) of the other investigated metabolites were at most 15% of that of MEHP. Parallel decreases in the plasma concentrations of DEHP, MEHP and the omega- and (omega-1) oxidized metabolites indicated that the elimination of DEHP was the rate-limiting step in the disposition of the metabolites. This was partly supported by the observation that the clearance of MEHP was higher than that of DEHP. Nonlinear increases in the AUCs of DEHP and MEHP indicated saturation in the formation as well as the elimination of the potentially toxic metabolite MEHP.

Acute tolerance to furosemide diuresis in humans. Pharmacokinetic-pharmacodynamic modeling.

Furosemide, 40 mg, was given to eight healthy volunteers as an i.v. dose and as oral doses (tablet and solution) with and without food intake. The urine and plasma were sampled frequently and analyzed on their content of furosemide (high-performance liquid chromatography). The urine flow and chloride excretion rate were used as measures of the effect. In spite of a 3-fold difference (28 vs. 9 mg/8 hr, P less than .001) in the cumulative urinary excretion of furosemide between i.v. and postprandial oral administration, no significant difference in the diuretic effect was found (2-2.2 liters/8 hr). The drug excretion-response curves showed parallel shifts depending on mode of administration of furosemide. Clockwise hysteresis, indicating acute tolerance development to the diuretic effect, was seen after the oral doses after food intake. This within-dose development of tolerance was modeled with an extended Hill equation. The tolerance development seems to have a near relationship to acute volume depletion (inadequate substitution of urine losses), probably activating some intrarenal mechanism for extracellular fluid volume preservation. Thus, the time course of furosemide excretion, as well as the degree of renal compensation, determine the renal sensitivity to furosemide. This has important implications for the proper design and interpretation of studies of the excretion-response relationship of diuretics.

Acute tolerance development to the diuretic effect of furosemide in the rat.

Furosemide was given to rats as five different i.v. bolus doses (2.5-100 mg kg-1), or as an i.v. infusion to a steady-state concentration in plasma of 14 micrograms ml-1. The urinary furosemide excretion rate (delta Ae/delta t) and the diuretic effect (volume of urine) were measured. A parallel shift in the excretion-response curve was seen as a fivefold increase in (delta Ae/delta t)50 [delta Ae/delta t) at half-maximal effect) between the i.v. bolus doses from 2.5 to 40 mg kg-1. The slope factor did not change. During infusion, a decrease in efficiency to 20 per cent of the initial value was seen. These results are indicative of an acute tolerance development to the diuretic effect of furosemide. Some intrarenal feedback inhibition mechanism might be involved, as the extracellular fluid volume seems to be of great importance to the effect obtained. The resulting effect can be compared with the influence of a competitive antagonist.

Implications of intraindividual variability in bioavailability studies of furosemide.

Intrasubject variation in bioavailability (rate and extent) and disposition of furosemide 40 mg was investigated using a repeated, randomized, double-blind cross-over study in 8 healthy subjects. Two generic tablet formulations (Lasix and Furix) and intravenous furosemide were compared on 6 separate days. Extensive intrasubject variability after oral administration was observed in AUC, mean absorption time (MAT) and urinary excretion. The variability (error variance) within the dosage forms was as large as that between the two generics. These variations most probably depended on the absorption process, since the repeated i.v. doses showed only marginal intrasubject variability. Absolute bioavailability was 56% for Lasix and 55% for Furix (AUC). The range was 20 to 84% between individuals and the maximal range within one individual was 20 to 61%. Confidence interval and Bayesian analysis showed a high probability of non-equivalence not only between but also within the generics when the separate cross-over experiments were analyzed (8 observations). When extending the analysis to 16 observations, bioequivalence was demonstrated for the two generic tablets. Rate of absorption, quantified as MAT, was 128 min for Lasix and 98 min for Furix (16 observations). Since MAT was significantly longer (p less than 0.001) than the mean residence time after the i.v. dose (57 min), absorption was evidently the rate-limiting step in the overall kinetics of oral furosemide. Intraindividual variation in absorption is a confounding factor in bioavailability studies of furosemide using limited numbers of subjects. This is important to consider when designing and evaluating bioavailability studies for drugs showing these variations.

Pharmacokinetics of furosemide in man after intravenous and oral administration. Application of moment analysis.

Furosemide 40 mg was administered to 8 healthy subjects as an i.v. bolus dose, as 1 tablet in the fasting state, and as 1 tablet and a solution after food intake. The i.v. data gave a total body clearance of 162 +/- 10.8 ml/min and a renal clearance of 117 +/- 11.3 ml/min; the volume of distribution at steady state was 8.3 +/- 0.61. Oral administration gave a bio-availability of the tablet (fasting) of 51%. Food intake slightly reduced the bioavailability, but not to a significant extent. There was no significant difference in availability between the tablet and the solution. Moment analysis gave a mean residence time after the i.v. dose, MRTi .v., of 51 +/- 1.5 min. The mean absorption times (MAT) for all oral doses were significantly longer than the MRTi .v., indicating absorption rate-limited kinetics of furosemide. On average, food delayed the absorption by 60 min. The MAT for the tablet in the postprandial state was significantly longer than for the solution, indicating dissolution rate-limited absorption of the tablet.

Dose-dependent pharmacokinetics of furosemide in the rat.

The pharmacokinetics of furosemide were investigated in the rat at doses of 10 and 40 mg kg-1 corresponding to doses of 80 and 320 mg given to humans based on body surface area. A three-compartment open model with renal excretion taking place from the shallow peripheral compartment gave the best fit to the data. The terminal half-life of furosemide was found to change from 29 min for the 10 mg kg-1 dose to 49 min for the 40 mg kg-1 dose. This change could be detected as a change in the apparent volume of distribution caused by decreased protein binding at increasing plasma concentrations of furosemide. The total plasma clearance did not change significantly although metabolic and renal clearance both changed. The renal clearance was found to be dependent on the free fraction of furosemide in plasma and thus increased with increasing plasma concentrations. The metabolic clearance decreased with increasing dose indicating a saturable metabolism of furosemide.

Analysis of nicotine as a trichloroethyl carbamate by gas chromatography with electron-capture detection.

Nicotine was subjected to reaction at 90 degrees with trichloroethyl chloroformate in the presence of pyridine to form a carbamate in which the pyrrolidine ring was opened. Upon heat treatment, this carbamate partially formed the corresponding olefin. About 10 pg could be detected with an electron-capture detector and 60 pg with an alkali flame-ionization detector. The extraction was studied with 14C-labelled nicotine. Methylene chloride was suitable for extraction from diluted plasma, whereas toluene containing 5% of heptafluorobutanol was used in a re-extraction step and also as the chloroformate reaction medium. Due to a nicotine blank the limit for quantitative determinations was 10 ng/ml in plasma (sample volume 1 ml). N-n-Propylnornicotine was used as an internal standard. The precision at the 30 ng/ml level was +/- 8.8% (n = 7).