Distribution of amiodarone in heart tissues following intrapericardial administration.

The distribution of amiodarone (A) in heart tissues of open chest anesthetized animals was studied at 20 and 60 min following the insertion of an isotonic solution of the drug into the pericardial sac (PS) in doses from 0.25 to 3 mg per kg body weight (BW) for pigs and after 60 min in doses of 3 to 6 mg for dogs. Most of the A absorbed by the myocardium was found in the subepicardium part of the left ventricular (LV) wall. For both species the percentage of drug absorbed by the myocardium was largely and inversely related to the dose given, while uptake by the atria was positively related to the dose and was higher than that in the subepicardial LV wall. Sixty minutes after i.v. administration of A (3.0 mg per kg BW) the drug was evenly distributed across the LV wall (16 microg per g wet tissue), which was significantly lower (p < 0.02) than that of subepicardal LV wall (30 microg) after intrapericardial (IP) administration. This study shows that satisfactory drug concentrations in predicted and specific distribution in the heart tissue were derived shortly after intrapericardial administration without measurable circulation in the blood.

The sarcolemmal Ca2+-ATPase of the ischemic-reperfused myocardium: protective effect of hypocalcemia on calmodulin-stimulated activity.

This study was designed to examine the role of calcium in the ischemia-induced changes of calmodulin-stimulated Ca2+-ATPase activity of heart sarcolemma of dogs subjected to coronary artery ligation (90 min) and reperfusion (30 min). This was attained by the application of systemic hemodialysis with low Ca2+ dialysate in six dogs (group A) and the comparison of the results with those obtained from animals subjected to normal Ca2+ hemodialysis (control group B, n=7). A very significant (p<0.001) decrease was found in the calmodulin-stimulated Ca2+-ATPase activity measured in the ischemic and non-ischemic parts of group B. This was associated with a decrease in the maximal velocity (v(max)) of the reaction of stimulation and an increase in the apparent Km for calmodulin. The kinetics of the calmodulin-stimulated Ca2+-ATPase also assessed in the presence of trifluoroperazine, a specific inhibitor for calmodulin binding, showed that the affinity for calmodulin was higher in the ischemic part of group A than of B, while v(max) was not substantially different. The above data may suggest that the inhibition of the calmodulin-stimulated Ca2+-ATPase produced by ischemia-reperfusion and its preservation under low Ca2+, are exerted at the calmodulin-binding site of the enzyme.

Rapid diminution of calcium in the myocardium by transfer to pericardial solutions. Myocardial dialysis.

A new method is described for the controlled and specific depletion of calcium from the vascularly perfused heart of experimental animals by means of dialysis, using a pericardial solution. A 30-40 ml isotonic phosphate buffer pH 7.3 with a low Ca2+ and high Mg2+ concentration (0.2 and 2.7 mM respectively) was inserted into the pericardial cavity of anaesthetized dogs and kept there for 10 or 60 min. The calcium content of the subendocardial and subepicardial halves of the left ventricular wall was similarly decreased to about 70% (P < 0.01) within 10 min and to 62% (P < 0.001) at 60 min, compared to that of hearts dialysed for 60 min in a standard solution of Ca2+ 1.2 mM and Mg2+ 1 mM. Calcium content of the myocardium dialysed with low Ca2+ and a standard Mg2+ solution decreased to only 75% (P < 0.01) at 60 min. Similar changes of calcium were measured in other parts of the heart. An increase in Ca2+ concentration in the pericardial solution was observed at the same time as a decrease in calcium in the myocardium. The increase in Ca2+ reached about 0.7 mM at 60 min, but decreased slightly, and finally, fell to 85% of pre-dialysis values at 60 min. It is concluded that this method of myocardial dialysis is effective in reducing myocardial calcium and is influenced by the duration of dialysis and the Mg2+ content of dialysate.

Distribution of lidocaine and digoxin in heart tissues and aorta following intrapericardial administration.

The distribution of lidocaine and digoxin in myocardial and aorta tissues of open chest anesthetized dogs was studied, following the administration of 30 ml phosphate buffer solution of the drugs in the pericardial cavity where it was kept for increasing time intervals. Transfer of lidocaine (15 or 30 mg) from the solution to myocardium was almost complete within 60 min, while only 50% of digoxin (2 or 50 micrograms) was removed, and this occurred during the first 30 min. Accordingly, the absorption rate of lidocaine by heart tissues increased with time up to 60 min while that of digoxin decreased with time. Absorption of digoxin by the atria and absorption of both drugs by intrapericardial aorta were higher than that of other heart tissues, between 20 and 60 min. At 30 and 60 min, lidocaine was evenly distributed across the LV wall while digoxin 50 micrograms was mainly concentrated subepicardially. On the contrary, i.v. administration of digoxin resulted in even distribution in the LV wall without preferential concentration in the atria. The uptake of both drugs by aorta was several times lower compared to heart tissues after i.v. administration. Drug concentrations in LV wall almost at therapeutic level, were derived from solution of low concentration of the drug in the pericardial cavity. It is concluded that intrapericardial administration of the drugs may be used when increased concentration of them is desired in specific areas of the heart and the aorta.

Effect of low calcium on high-energy phosphates and sarcolemmal Na+/K(+)-ATPase in the infarcted-reperfused heart.

This study was undertaken to compare the effect of low to normal serum calcium on biochemical parameters in the myocardium of dogs subjected to 90 min of coronary artery ligation followed by 30 min reperfusion. The accumulation of calcium, the decrease of adenosine triphosphate (ATP) and creatine phosphate (CP) and the inhibition of sarcolemmal ouabain-sensitive Na+/K(+)-ATPase which are prominent findings in the ischemic-reperfused myocardium, were studied under normal and low serum Ca produced by normal and modified hemodialysis (HD). The results showed a lower accumulation of Ca (P less than 0.002) in the ligated-reperfused myocardium of dogs subjected to low-calcium HD. In the same group of animals ATP was protected to some extent while CP was completely preserved. This may indicate that during reperfusion with low Ca, restored ATP is further utilized for CP regeneration. The activity of Na+/K(+)-ATPase was within normal values in the ligated-reperfused myocardium of the low-calcium group. The significantly (P less than 0.001) negative correlation between tissue calcium concentration and Na+/K(+)-ATPase activity under various conditions examined, provided additional evidence that low calcium is a protective factor of the enzyme activity during ischemia and reperfusion.

Amiodarone--application and clinical pharmacology in atrial fibrillation and other arrhythmias.

Forty patients with atrial fibrillation (AF), 23 patients with ventricular extrasystoles (VES), and 11 patients with various arrhythmias (VA) were treated with amiodarone (0.2-0.6 g/day). Suppression of arrhythmia was 67.5% in AF, 78.2% in VES, and 81.8% in others with VA. Median age of converted patients was higher than that of nonconverted. The duration of AF before treatment was inversely related to drug efficacy. Average time needed for conversion was 6-8 days of treatment. Plasma amiodarone concentration at the day of conversion did not differ from that of nonconverted patients. Amiodarone concentration levels off after the 8th day of treatment, whereas that of the metabolite increases with time of treatment. Biologic half-life of plasma amiodarone after discontinuation of treatment varied, but was higher than 4 days. The percent of decline of the metabolite concentration was lower than that of the parent drug.

Effect of hypocalcemia on isoproterenol induced cardiotoxicity in dogs.

The effect of serum calcium on the myocardial damages produced by isoproterenol was studied in the dog. Hemodialysis for 80 min in the absence of calcium was used to alter serum calcium concentration in seven experimental animals. The same number of animals were dialysed in the presence of calcium and were used as controls. After hemodialysis all animals were infused with isoproterenol 2.0 micrograms/kg/min for 4 hours. The myocardial damage was assessed by comparing serial measurements of the serum cardiac enzyme activities CK and CK-MB and the electrocardiographic findings of the two groups, before, during and after isoproterenol infusion. Serum calcium decreased significantly after dialysis only in the experimental group (1.15 vs 2.19 mmol X L-1). Total CK activities of the experimental group during and after isoproterenol infusion were 2 to 3 times lower than in the controls (190-360 vs 410-1370 IU X L-1). Changes of the CK-MB isoenzyme activities were more profound, these were detectable and measured only in 4 of the experimental animals and in much lower activities than in the controls (25-61 vs 45-445 IU X L-1). A positive correlation was found (r = 0.673, p less than 0.05) between the highest value of CK-MB of both groups and the serum calcium concentration after hemodialysis. In accordance to the differences of the biochemical estimates the effect of serum calcium on the cardiotoxic effect of isoproterenol has been further emphasized by the electrocardiographic findings. Thus arrhythmias, negative T-waves and significant ST segment depression were observed only in one experimental animal. In contrast these electrocardiographic disturbances were common and marked findings in all but one of the control animals. In both groups these electrocardiographic findings were reversed one to three hours after the termination of the isoproterenol infusion. Myocardial calcium at the end of the experiment was lower in the experimentals compared to the controls (0.23 vs 0.30 mmol X kg-1 wet weight). On the contrary magnesium concentration increased respectively. It is concluded that low serum calcium has a protective effect against the cardiotoxic action of isoproterenol. This protective action may be relevant to the decreased calcium and increased magnesium of the heart of the animals hemodialysed in the absence of calcium.

Pharmacokinetics of amiodarone after intravenous and oral administration.

Pharmacokinetic studies were conducted in four subjects who received 150 mg amiodarone i.v., in nine subjects who received 200 or 400 mg amiodarone p.o., and in four subjects who received the drug by both routes of administration. Amiodarone concentrations were determined by high pressure liquid chromatography. Aminodarone levels after i.v. administration, with one exception, declined biphasically according to a two-compartment model. The decline appeared to be monophasic after p.o. administration with one exception. Plasma levels after p.o. administration varied considerably between subjects. The biologic half-life after p.o. administration (mean 17.1 h +/- S.D.) was higher than that calculated for the second phase after i.v. administration (mean 4.3 h +/- 0.98 S.D.). Total clearance rate during the second phase was within the range 125-288 ml/min after i.v. and 114-473 ml/min after p.o. administration. The apparent volume of distribution in the central compartment after i.v. administration was found to be about 17.0 l and that calculated for the second elimination phase, 77 l, possibly indicating a concentration of the drug in the tissues. The systemic availability of amiodarone varied from 23 to 50% as determined on the basis of of the ratios of areas under p.o. and i.v. serum concentration curves for four subjects.

Studies on glycosphingolipid biosynthesis by lectin-stimulated human lymphocytes.

1. Human peripheral lymphocytes, normal and stimulated by phytohemagglutinin or concanavalin A. were investigated with respect to their ability to biosynthesize neutral and acidic glycosphingolipids from D-[U-14C]glucose, D-[U-14C]galactose and D-[U-14C]glucosamine as precursors. 2. Galactose and glucosamie are taken up selectively, in the presence of excessive glucose concentrations. Labeling of total neutral glycosphingolipids from D-[U-14]galactose in normal cells decreases after the first 6 h while in stimulated cells there is a fourfold and a sevenfold increase after 6 and 12 h of incubation respectively. Under similar conditions stimulation with concanavalin A gives a fourfold increase after 12 h of incubation. 3. Analysis of individual glycosphingolipids biosynthesized from [U-14C]galactose shows that lactosylceramide is the major radioactive neutral glycosphingolipid and that stimulation by phytohemagglutinin yields an almost sevenfold and 13-fold increase in the radioactivity incorporated within 6 and 12 h of incubation respectively. Glucosylceramide shows about an eightfold increase, globotetraosylceramide a threefold increase and globotriaosylceramide a fourfold increase. The rate of incorporation into glucosylceramide of stimulated lymphocytes declines after 6 h of incubation, accompanied by a concomitant increase of incorporation into lactosylceramide. 4. At 12 h of incubation the ratios of radioactivities incorporated into neutral glycosphingolipids of phytohemagglutinin-stimulated cells compared to normal cells were 1.0 for D-[U-14C]glucose 7.0 for D-[U-14C]galactose and 2.5 for D-[U-14C]glucosamine. Respective ratios for lactosylceramide are 1.0 for [U-14C]glucose and 13.0 for [U-14C]galactose. These differences did not arise from changes of the uptake of the glycosyl precursor by the cell due to stimulation. 5. Incorporation of D-[U-14C]galactose into II3-N-acetylneuraminosyllactosylceramide by cells stimulated by phytohemagglutinin and concanavalin A is enhanced 14-times and 15-times respectively. With [U-14C]glucosamine as precursor, this increase in the labeling is much more impressive, 80-fold after 12 h of incubation by the phytohemagglutinin-stimulated lymphocytes. Neuraminidase treatment and gas radiochromatographic analysis of the labeled compound derived from [U-14C]-galactose as precursor indicate that 89% of the radioactivity was incorporated into the glucosyl and galactosyl moieties, in a ratio 1:1. With [U-14C]glucosamine as precursor, a selective labeling of the sialyl moiety of the II3-N-acetylneuraminosyllactosylceramide was indicated. 6. The pattern of complex gangliosides (more complex than II3-N-acetylneuraminosyllactosylceramide) which are biosynthesized after phytohemagglutinin stimulation of the cells, show no significant differences when compared to the patterns obtained from normal cells.

Isolation and partial characterization of the neutral glycosphingolipids and gangliosides of the human heart.

The glycosphingolipids (GSL) of the human heart muscle have been isolated from total lipids by column and thin layer chromatography and their sugars and fatty acids analyzed by gas liquid chromatography. Hearts from traffic victims were obtained at autopsy between 12 and 16 hr after death and dissected into parts (left and right ventricular walls, intraventricular septum and papillary muscle). The neutral GSL content for those parts of the hearts of two males aged 22 and one female aged 14 ranged from about 90 to 160 nmoles/g wet weight. Trihexosyl ceramide and globoside were the most abundant neutral GSL. Total ganglioside content was about 50 nmoles/g wet weight, and the most abundant gangliosides were partially characterized as GM3 and GM1; other mono-, di- and trisialogangliosides were also present. Differences in the content and composition of neutral GSL and gangliosides between the heart and other human tissues are discussed. It is concluded that the patterns of these two GSL fractions of the heart are more complex than those of the extraneural human tissues.

Changes in rat brain gangliosides following active avoidance conditioning.

Brain gangliosides of rats trained in a conditioned avoidance Sidman task and undisturbed rats in their cages were studied. The (14C) acetate was injected intracerebrally seven days before the starting of 30 days training. Thirty-seven days after injection all rats were killed and ganglioside fractions were isolated from neocortex, hippocampus, brain stem, cerebellum and residual cerebral tissue of each one brain. Trained rats had higher levels of (14C)-labeled polysialogangliosides (G1, G2, G3) in hippocampus and neocortex than the controls. Regarding the rest of the brain areas, a significant increase of G2 in the residual cerebral tissue of the trained as compared with the controls was found. The results suggest that the sialic acid rich gangliosides of only certain parts of the brain are affected by the Sidman avoidance conditioning of the animals.

Lipid composition of lymphocyte plasma membrane from pig mesenteric lymph node.

1. The lipid fraction of the plasma membrane of pig mesenteric lymph-node lymphocytes contained primarily (94%) neutral lipids and phospholipids in about equal weights. The remianing lipid comprised glycosphingolipids (1.8%), gangliosides (o.27%)and probably ceramides (1.3%). The major phospholipid was phosphatidylcholine (46% of the total), and mono- and tri-hexosylceramides accounted for 72% of the glycosphingolipids. Haematoside was distributed between the glycosphingolipid and ganglioside fractions. The major ganglioside was monosialoganglioside. About 90% of the sialic acid was N-glycollylated. 2. A comparision of the lipid composition of the plasma-membrane fraction with that of the initial lymph-node homogenate showed that the purified membrane contained increased proportions of phospholipids, especially sphingomyelin, glycosphingolipids and gangliosides. 3. Fatty acid analyses showed that the membrane phosphatidylcholine was rich in palmitic acid, that the sphingomeyelin and phosphatidylethanolamine were high in myristic acid and that the glycosphingolipids were rich in oleic acid.

R-mediated beta-lactamases and episomal resistance to the beta-lactam drugs in different bacterial hosts.

Ten penicillinase plasmids of varying taxonomic origin were studied after transfer to a variety of bacterial hosts. Nine of the ten plasmids specified enzymes with the following identical, or very similar, properties: substrate profile, molecular weight, susceptibility to heat and inhibitors, and electrophoretic mobility, i.e., TEM-like enzymes. The tenth R-mediated beta-lactamase was a cephalosporinase. Plasmids with TEM-like enzymes mediated resistance patterns identical towards the beta-lactam drugs, whereas the resistance pattern of the cephalosporinase plasmid was distinctly different. Expression of enzyme and resistance had a dual R-factor and host specificity. Escherichia coli K-12 and Salmonella typhi constituted one group of the same R-factor phenotype expressions. Most, but not all, penicillinase plasmids exhibited in Proteus PM1 a considerably lower order of beta-lactamase activity and an even lower order of resistance to the beta-lactam drugs than the previous two hosts. This difference was most pronounced for the resistance to carbenicillin, which was mediated by the plasmids specifying the synthesis of TEM-like enzymes. Release by osmotic shock was complete in the host E. coli K-12 for the TEM-like enzymes, but was lower for the cephalosporinase and minimal or negative in the PM1 host. Crypticity factor for benzylpenicillin, ampicillin, and carbenicillin was not related to the increase in resistance mediated by the penicillinase plasmids in both K-12 and PM1 hosts. Inoculum size effects for the penicillins and 6-aminopenicillanic acid were higher in PM1 than in K-12 R(+) cultures. The expression of penicillinase plasmids in wild-type bacteria was strain specific and not species specific. For two plasmids of different phenotypes for beta-lactamase activity (and resistance) in K-12 and PM1 hosts, a positive correlation was found between their phenotype and the relative amount of episomal deoxyribonucleic acid, as detected by ethidium bromide density gradient centrifugation. This is interpreted as indicating differences in the mode of replication of the plasmids in the two hosts.