John P. Kaltenbach

Electrolyte Alterations in Acute Myocardial Ischemic Injury

The electrolyte shifts occurring in myocardial cells irreversibly injured by ischemia were compared in two groups of dogs; one given 40 minutes of transient ischemia followed by 20, 50, 80, and 200 minutes of restored coronary arterial flow and the other 30, 60, 90, 130, and 240 minutes of permanent ischemia. Each interval of restored arterial flow in the transient ischemia group was associated with marked changes in the electrolyte content of the injured myocardium. Sodium, chloride, and water were increased and potassium and phosphorus were decreased. These changes occurred much more quickly in dogs exposed to transient ischemia than in dogs with comparable periods of permanent ischemia. These data indicate that objective chemical evidence of the death of myocardial cells develops slowly in areas of permanent ischemia primarily because of the reduced flow of blood through the ischemic injured tissue.

Oxygen-induced enzyme release: Early events and a proposed mechanism

Abstract Rat hearts perfused for 60 min with hypoxic Krebs-Henseleit medium were fixed and examined by light and electron microscopy at 0, 0.5, 1, 2, 5 and 15 min after reoxygenation. The earliest structural changes seen at 0.5 to 2 min were contracture of myocardial cells with separation of sarcomere attachments to intercalated discs and Z-bands. These separations were followed by focal, and later, diffuse mitochondrial and cytoplasmic swelling. Creatine phosphokinase release from hearts could be detected by 0.5 min, but did not peak until 2 min after reoxygenation. The observed temporal sequence of events suggests that contracture of cells precedes enzyme release and massive cell swelling. It is postulated that when oxygen-supported contracture occurs, stretching and rupture of cell sarcolemmal membranes occurs in dyskinetic regions of cells left unsupported by sarcomere detachments.

Anoxia, calcium and contracture as mediators of myocardial enzyme release ☆

Abstract The effects of anoxia and calcium-free perfusion on enzyme release, were studied in perfused rat hearts, at 37°C. In oxygenated hearts, calcium-free medium caused detachment of cells at intercalated discs, but no cell separation or enzyme release. Sixty minutes of anoxia with calcium-free medium caused striking cell separations, contracture of sarcomeres and enzyme release. Anoxia, followed by calcium-free medium, produced a sudden, energy-independent enzyme release with contracture and cell separations. Contraction bands as well as cell separations, were present in anoxic hearts reoxygenated in the absence of calcium. It is proposed that calcium-free media causes loss of integrity of intercalated disc junctions and contraction, and anoxic contracture pulls cells apart at weakened cell junctions, mediating membrane damage and enzyme release.

Renal tubular necrosis induced by compounds structurally related to d-serine

Abstract d -serine is nephrotoxic in rats causing acute tubular necrosis of the straight segment of the proximal tubule. In an effort to determine the mechanism of this injury, the nephrotoxic effect of eighteen compounds structurally related to d -serine was investigated. Necrosis of the proximal straight tubules of rat kidneys was not observed with compounds wherein the hydroxyl or amino group of d -serine was blocked or eliminated, a methyl group was on the same carbon atom as the hydroxyl or amino group, the hydroxyl and amino groups were reversed, or the carbon chain lengthened. Only dl -2,3-diaminopropionic acid, other than d -serine itself, consistently induced proximal straight tubular necrosis. The l -isomer was without effect. Our results suggest that the nephrotoxicity observed with d -serine and dl -diaminopropionic acid is related to their selective absorption, based, within narrow limits, upon functional groups, size, and configuration of the molecule.

Oxygen-induced enzyme release. Assessment of mitochondrial function in anoxic myocardial injury and effects of the mitochondrial uncoupling agent 2,4-dinitrophenol (DNP)

Abstract Hypoxic injury to isolated-perfused rat hearts resulted in progressive loss of mitochondrial function, as assessed by polarographic techniques. Decreased mitochondrial respiration could be detected as early as 15 min after beginning hypoxic-substrate-free perfusion and by 75 min little or no functional capacity could be detected. Control studies using mixtures of mitochondria from control and damaged hearts suggested that polarographic techniques tend to overestimate mitochondrial injury. In hearts injured by hypoxia, mitochondrial damage was heterogenous and there was a good linear correlation between the respiratory control ratios of mitochondrial suspensions and the percent damaged mitochondria in the suspensions, as determined by electron microscopy. Respiratory control ratios approached one when only 60 to 70% of mitochondria appeared morphologically damaged. The evidence suggested that significant mitochondrial function was retained in hearts after 60 min of hypoxic perfusion, a time when oxygen-induced enzyme release was nearly maximal. In similarly perfused hearts, both oxygen-induced enzyme release and associated anomalous cellular contracture were reduced or completely inhibited by 0.1 or 1 m m 2,4-dinitrophenol (DNP), but not by 0.04 m m DNP. In intact hearts, 1 m m DNP was required to produce biological effects on cellular morphology and enzyme release equivalent to those produced by either hypoxia or cyanide. These results provide evidence that both oxygen-induced enzyme release and cellular contractures are energy dependent events mediated by resumption of mitochondrial oxidative phosphorylation.

Immunological evidence of repressed catalase synthesis in livers of tumor-bearing mice.

Abstract A quantitative immunochemical procedure for the determination of hepatic catalase in livers of mice is described. By employing this method, the data obtained suggest a sharp and marked diminution of synthesis of catalase in livers of mice with an advanced growth of Ehrlich ascites tumor. The magnitude of depression in the activity of liver catalase is related to the reduction in absolute concentration of the enzyme protein in liver homogenates of tumor-bearing animals.

Protection from irreversible hypoxic injury by potassium cardioplegia and hypothermia: Effects on contracture, morphology and O2-Enzyme release

Abstract Isolated rat hearts were perfused at temperatures from 40 to 30°C in the presence and absence of 16 m m potassium cardioplegia, with hypoxic substrate-free Krebs-Henseleit medium. During hypoxia the development of contracture was monitored with an intraventricular balloon. After intervals from 10 to 300 min of hypoxia hearts were reoxygenated at 37°C and the released creatine kinase activity (CK) was measured for a 15 min interval. At the end of experiments, the percentage of injured cells was estimated by light microscopy. Both hypothermia and cardioplegia delayed the onset and reduced the rate of development of contracture. The effects of cardioplegia and hypothermia were additive. Irreversible injury was delayed proportionally with the delay in full contracture development. Hypothermia caused a progressive decrease in the rate of progression of injury. Cardioplegia also reduced the slopes of injury curves, but its major effect was to delay the onset of injury. Between 40 to 30°C there was a nearly linear decrease in the slopes of injury curves from a rate of 6.6% per min. at 40°C to 0.85% per min. at 30°C. Estimates of injury by CK release and morphology were similar. Severe hypoxic injury was associated with marked reductions in coronary flow rates to about 40% of control flows. In hypoxic hearts at 37°C vascular infusion of colloid carbon demonstrated subendocardial zones of severe ischemia ranging from 10 to 40% of heart ventricular masses. There was no apparent correlation between coronary flow rates and the size of observed perfusion defects. It is concluded that myocardial protection occurs as the combined effect of delaying the onset of injury and slowing its progression after initiation. The proportion of cells in a heart showing morphologic evidence of injury correlated with the observed enzyme release. Injury appeared to progress in quantum steps as individual cells made the transition from reversible to irreversible injury. Reductions in slopes of injury curves with protection could be explained as a slowing of the rate of energy depletion in a heterogenous cell population and a spreading of the time interval over which cells reached a critical level of energy depletion. The existence of perfusion defects and concomitant ischemia may limit the isolated perfused rat heart as an experimental model of hypoxic cell injury.

Compounds protective against renal tubular necrosis induced by d-serine and d-2,3-diaminopropionic acid in the rat☆

Abstract d -Serine and d -2,3-diaminopropionic acid ( d -DAPA) induce necrosis of the epithelial cells of the proximal straight tubules of the rat kidney. Nineteen compounds with chemical structures and/or functional groups similar to d -serine or d -DAPA were tested for their protective effect against the induction of this injury. The acute tubular necrosis inducible by d -serine and d -DAPA was prevented by d -alanine, d -threonine, d -homoserine, dl -α-methyl serine, β-hydroxy- dl -leucine, and α-aminoisobutyric acid. In addition, protection from necrosis due to d -DAPA, but not that due to d -serine, was found with l -threonine, l -serine, l -2,3-diaminopropionic acid, and 3-hydroxypropionic acid. Little or no protective effect against d -serine of d -DAPA was afforded by l -alanine, β-alanine, dl -isoserine, dl -3-hydroxybutyric acid, l- or dl -lactic acid, 2-amino-2-methyl-1-propanol, taurine, and isethionic acid. The compounds that protected against d -serine-induced necrosis had in common the d -configuration (except for aminoisobutyric acid), the α-amino group, and the carboxyl group. The structural characteristics and chirality of the protective agents against d -DAPA were more varied and less definable than for d -serine. d -Alanine, dl -α-methyl serine, and α-aminoisobutyric acid protected on a mole-for-mole basis against d -serine-induced necrosis. d -Alanine was protective against d -serine even when given 30 min after d -serine administration. These results suggest that protection may be due to competition with d -serine or d -DAPA for a cellular or transport binding site.

Effects of hyperosmolar solutions of polyethylene glycol, dextran or mannitol on enzyme release from perfused rat hearts.

Abstract The effects of polyethylene glycol (PEG), mannitol and dextran on the osmotic properties of solutions, and the effects of hyperosmolar solutions of these oncotic agents on swelling of tissue slices and creatine phosphokinase (CPK) release from Langendorf-perfused rat hearts were studied under oxygenated and anoxic conditions and after reoxygenation following an anoxic interval. Mannitol solutions exhibited nearly ideal osmotic behavior, while both solutions of dextran and PEG showed marked deviations from ideal behavior. Hypertonic dextran and PEG solutions prevented both anoxic and cold-induced swelling of tissue, while mannitol only slightly reduced tissue swelling. Anoxic enzyme release was reduced by PEG, but not by dextran. Hypertonic perfusion media containing either mannitol or dextran, but not PEG, caused cell damage and CPK release from rat hearts during oxygenated perfusions. All these oncotic agents reduced both cell swelling and oxygen-induced CPK release, suggesting that cell swelling may be important in mediating this injury. The role of cell swelling in anoxic enzyme release cannot be determined from these experiments because of the possible superimposed cytotoxic effects of dextran and mannitol.

Murine immunoglobin response to sterile talc injection

Abstract Mice were injected subcutaneously with sterile talc, and the changes in serum IgG, IgM, and IgA were investigated at various time intervals. Three talc-injection schedules were used: a single injection of talc; a double injection of talc; and two injections of talc at different times. The results showed that: (1) a single injection of talc resulted in an increase in serum IgM at 24 hr, and an increase in IgG at 24–48 hr; (2) a double injection of talc did not result in a significant difference in immunoglobulin levels from those observed after a single injection; (3) two injections of talc given at different times did not result in a response greater than that observed following a single injection of talc. No changes from control values were observed in IgA. No evidence for the presence of an antigen was found.