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A toxic extent has been isolated and partially purified from burnt human and mouse skin and also from sera of severely burnt patients, which causes disturbances of energy metabolism and decreased synthesis rates for glucose and urea in the perfused rat liver. Enzymatically isolated hepatocytes from rat livers were used to study the toxic effects on hormonal sensitivity, synthetic functions and ultrastructure of the cells. A decreased synthesis of urea and glycogen was found in cells from rats treated 5 days before with "toxin" and in cells, which were directly incubated with the toxic factor. Glucagon increased urea synthesis in normal cells by 33%, and a decrease of 25% was caused by insulin. Cells of rats treated with the nontoxic precursor of the toxic factor from normal skin were similar, while those treated with "toxin" produced less urea and did not react to glucagon or insulin. Glycogen synthesis was reduced in cells directly incubated with the "toxin", however, the hormonal effects were still observed. Surface alterations of "toxin" treated cells and cells of "toxin" treated rats were found by scanning electronmicroscopy. These findings provide evidence of a direct cytotoxic effect of the toxic factor from burnt skin. It is proposed that the "toxin" acts on the cellular membrane with destruction of surface and receptorproteins.

Early and adequate shock treatment combined with improved systemic and local therapy practically eliminated primary death of patients with severe burns. In contrast the late mortality, i.e. the fatal outcome during the "burn disease" has not been significantly improved during the last decade. A burn toxin has been discussed as reason of this burn disease. Such a toxic factor has been isolated from burnt mouse and human skin and from the serum of burnt patients as well. Electron microscopic studies in rats revealed similar and comparable mitochondrial alterations of hepatocytes after either a sublethal controlled burn injury or an i.p. application of an equivalent dose of this cutaneous burn toxin. These alterations were dose-dependent. Studies of the liver metabolism of these rats (i.e. gluconeogenesis, urea synthesis and energy metabolism) suggested an inhibited oxidative phosphorylation. Incubation of enzymatically isolated hepatocytes with toxin demonstrated a direct cytotoxic effect of the burn toxin by scanning electron microscopy. Glycogen synthesis and urea synthesis were significantly reduced, hormonal sensibility was partially abolished. These results suggest a disturbance of the cellular energy metabolism via a cell membrane damage. This could be the reason of the decreased host defence of burned patients which causes the fatal outcome in severe burns. Therefore, elimination of burn toxin should be an important point in burn treatment.

Studies on isolated perfused rat livers 5 days after either a sublethal burn or an i.p. injection of human/mouse burn toxin showed a significant inhibition of the glucose/urea synthesis and the ATP production concomitantly with ultrastructural mitochondrial damages. A direct specific effect of these burn toxins on enzymatically isolated liver parenchyma cells was found either after direct incubation of the isolated cells with the compound or 5 days after injection of the toxin to the animals followed by the isolation of the cells. Control experiments were performed with the "native" non-toxic precursor from normal skin. Liver cells of rats pretreated with the toxin showed an 100% increase of the amino-acid release while this increase was 70% after direct toxin incubation. Glycogen synthesis from lactate, alanin and fructose was significantly decreased in both toxin groups while the glucose synthesis was not altered. The degree of the inhibition of the glycogen synthesis was directly correlated to the number of ATP-dependent metabolic steps. A disturbance of the oxygen transfer system by structural damages of the mitochondria seems to be the basic mechanism for these specific metabolic alterations due to ultrastructural mitochondrial damages.

Ultrastructural alterations of the liver were examined on the 1st, 2nd, 3rd, 5th, 7th and 14th day after a standard burn in mice. Our results can be divided pathogenetically into two groups: 1. Alterations explained by the primary thermal injury including electron-optically empty vacuoles and sinusendothelium destruction. 2. Alteration due to the influence of a specific burn toxin including mitochondrial changes and the formation of autophagic vacuoles. Evidence of this distinction was produced by a second investigation. Burn toxin isolated from the serum of severely burned patients was injected intraperitoneally (15 mg) into 4 mice. The livers were examined by electron microscopy on the 2nd, 4th, 7th and 14th day. Alterations described in 2 above were not observed in any case of this investigation series. The nature and timing of liver alterations were similar to those described in 2 above. In none of the cases were changes similar to those in 1 above observed.

The existence of a burn toxin which could be responsible for the late burn disease has become increasingly accepted. The present study investigates both metabolism and ultrastructure of isolated rat hepatocytes both under the influence of a burn toxin isolated from burnt mouse and human skin and of its native nontoxic precursor. Cells from rats treated with toxin systematically were also investigated. The cells directly incubated with toxin showed no alterations of gluconeogenesis, but a reduced urea--and glycogensynthesis from most precursors used. Cells of pretreated rats were reduced in all functions and showed more distinct ultrastructural damage, while those incubated directly were significantly more altered. The results prove a direct toxic effect of a burn toxin on isolated liver cells.

Two different peptides have been purified from human liver, similar to those previously reported (Schoenenberger, G.A., and Wacker, W.E.C. (1966) Biochemistry 5, 1375--1379) to be present in human urine, which may serve as metabolic regulators of lactate dehydrogenase (EC 1.1.1.27) isoenzymes (LDH-M4 = muscle type; LDH-H4 = heart type). By trichloroacetic acid precipitation, ultrafiltration, Sephadex G-25 and Bio-Gel P-2 columns, affinity chromatography on immobilized LDH-isozymes and HPLC two peptides which differed with respect to molecular weight, retention on the affinity columns and amino acid composition were isolated. No effect was observed when native, tetrameric lactate dehydrogenase was incubated with these peptides. However, when lactate dehydrogenase was dissociated to monomers at low pH and allowed to reassociate by adjusting the pH to 7.5 complete inhibition of the reactivation occurred when the inhibitors were incubated together with respective reassociating monomeric isozymes. The two peptides showed no cross-specificity, i.e. each peptide exhibited inhibitory activity only on one of the two isozymes LDH-M4 or LDH-H4. From the amino acid analyses, gel filtrations and PAGE + SDS, molecular weights of 1800 for the M4 and approximately 2700 for the H4 inhibitor were calculated. An apparent Ki of approximately 3 X 10(-5) mM for the H4 and approximately 7 X 10(-5) mM for the H4 inhibitor was estimated. The interaction of the inhibitors with the enzyme system showed strong cooperativity with Hill coefficients of 2.9 (LDH-M4-specific) and 2.4 (LDH-H4-specific). Mathematical modelling of the reassociation and reactivation of lactate dehydrogenase and its specific inhibition by the peptides led to the conclusion that the peptides react with monomers, dimers or a transition state during the tetramerisation process. kappa 1 for the dimerisation step of M4 = 2.0 X 10(5) M-1 . S-1 and of H4 = 8.2 X 10(4) M-1 . S-1; kappa 2 for the tetramerisation step of M4 = 2.8 X 10(5) M-1 . S-1 and of H4 = 1.2 X 10(5) . M-1 S-1, were calculated, the second step still being the faster one (Rudolf, R. and Jaenicke, R. (1976) Eur. J Biochem. 63, 409--417).

The catalytic activity of the LDH-isoenzymes depends on their tetrameric structure. Low pH or other denaturants leads to dissociation into monomers and to the loss of the specific activity. After removal of the denaturing conditions reassociation and reactivation occur spontaneously. Neither NADH nor NAD+ shows a significant effect on the reactivation. We have isolated two different peptides which isoenzyme specifically inhibit the reactivation of dissociated LDH. Inhibition was abolished by treating with proteases. Additionally, NAD+ and NADH were found to be antagonists of the inhibitors. The heart-type enzyme-inhibitor system is especially susceptible for NADH whereas NAD+ affects the inhibition only slightly. The muscle-type system shows the opposite behavior, e.g., the completely inhibited system can be fully reactivated by NAD+ but not by NADH. These findings together with first kinetic studies suggest a possible specific regulatory function of these peptides.