Biochemical Lesion of Diphtheria Toxin in the Heart*

Abstract

Clinical and experimental investigations have shown that the myocardium is highly susceptible to the action of diphtheria toxin (1, 2). Both in man and the guinea pig, one of the earliest alterations in the heart muscle exposed to this toxin is fatty degeneration (2, 3). This morphological observation along with recently acquired evidence that fatty acids are a major metabolic substrate of the heart (4) suggested that the toxin may exert its effect on the myocardium by interfering with fatty acid oxidation.Experiments were designed to test this hypothesis by ascertaining the effect of diphtheria toxin on 1) the capacity of the myocardium to oxidize long-chain fatty acids, 2) the integrity of component parts of the fatty acid oxidation pathway in the heart, and 3) the concentration of myocardial carnitine (DL-7-trimethylamino-P-hydroxybutyrate), a compound known to effect a stimulation of long-chain fatty acid oxidation in the heart (5). MethodsWhite, male guinea pigs weighing 250 to 300 g were used. A standard diet consisting of 50% Purina rabbit chow and 50% oats supplemented with fresh cabbage was used except as indicated. The experimental animals were divided into 4 groups as follows: a) Diphtheria-toxin 1 animals were injected with toxin subcutaneously, the dose being adjusted so that fatty degeneration of the myocardium could be produced regularly in surviving animals within 5 days after treatment; b) diphtheria-antitoxin 1 animals were injected intraperitoneally with 500 U of antitoxin; c) diphtheriatoxin and -antitoxin animals were injected with 500 U of * Submitted for publication August 27, 1963; accepted November 29, 1963. Presented in part at the Fifty-fifth Annual Meeting of the American Society for Clinical Investigation, Atlantic City, N. J., April 1963. This work was supported by U. S. Public Health Service grants H-7061, HE 07780-01, and 5TI GM-516-03.1 Diphtheria toxin, lot #706396, and diphtheria antitoxin, lot #G-138-B, were supplied by Eli Lilly and Co., Indianapolis, Ind.antitoxin 24 hours before administration of the toxin; and d) fasting animals were deprived of food and given water ad libitum. Each experimental animal used for an enzymatic assay was tested simultaneously with a paired untreated mate as a control. For all other determinations, groups of experimental animals were compared with groups of controls.Five to six days after injection of the toxin or the beginning of the fast period, the guinea pigs were sacrificed by a blow on the head. The entire heart was removed immediately, the great vessels were trimmed off at their origin, and the cardiac cavities were freed of blood. A transventricular block of myocardium was placed in 10% neutral, buffered formalin solution and kept at room temperature for at least 24 hours. Frozen sections, 14-As thick, were prepared and stained as follows: a) hematoxylin and eosin, b) oil-red 0 dissolved in acetone alcohol, and c) oil-red 0 followed by hematoxylin. Histological examination was performed on all hearts except for those used for quanti...

Defective lipid metabolism in the failing heart

“…The capacity of left ventricular homogenates to oxidize selected substrates was assessed by collection of "4CO2 from "4C-labeled substrate as previously described (18). Each constricted and sham animal used for an enzymatic assay was tested simultaneously with an untreated mate as a control.…”

The effect of thyroxine on lipid and carbohdrate metabolism in the heart.

“…Assays of palmitate, pyruvate, and glucose oxidations were carried out as previously described (12). Since the myocardial concentrations of lipid in the treated and control animals were not the same, the endogenous pools of FFA in the hearts were ascertained before the addition of radioactive palmitate.…”

Lipid metabolism in the newborn heart.

“…The capacity of the cardiac homogenates to oxidize selected substrates was assessed by collection of C1402 from C'-labeled substrate as previously described (11). The specific activities of the palmitate and the glucose in the reaction mixtures were estimated as follows: the concentration of free fatty acid in the myocardium was determined by the method of Amenta (12), and that of glucose by the procedure of Huggett and Nixon (13 After the C1402 produced in the palmitate assay had been collected, the lipids in the reaction mixture were extracted as previously described (14).…”

“…In vivo, however, the low carnitine concentration may be equal in significance to or more significant than the low transferase activity in limiting the oxidation rate of long chain fatty acids in the newborn heart. Evidence has been obtained that suggests that lack of carnitine alone can limit the rate of long chain fatty acid oxidation in the heart (11). Further analysis of this problem in the newborn heart would require manipulation of the level of activity of the transferase in the assay of palmitate oxidation.…”