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The mechanism of the tetrazolium reaction in identifying experimental myocardial infarction

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Tetrazolium salts (NBT) stain normal myocardium whereas infarcts are not stained. We tried to elucidate the staining mechanism which discriminates normal from infarcted canine myocardium. The left anterior descending coronary artery (LAD) was occluded in dogs for between 4 and 32 h. The activities of four different tissue dehydrogenases were measured after 4, 8, 16, and 32 h of ischaemia. Nicotinamide adenine dinucleotides (NAD, NADH, NADPH) were determined in needle biopsies taken from the ischaemic region 1/2, 1, 11/2, 2 and 4 h after occlusion of the LAD. In another set of experiments the NBT stain was altered by the addition of NADH, NAD, NADPH, NADP, succinate, lactate and phenazine methosulfate respectively and the effect of the added substances on the previously nonstained infarcts was examined. We further compared histochemically determined infarct size to the ultrastructural extent of infarcts. Activities of the tissue dehydrogenases did not change after 4 h of ischaemia, although the NBT stain revealed a large infarction. At that time total NAD, the sum of NAD+NADH, had decreased from about 600 pmoles/mg tissue to about 200 pmoles/mg tissue and addition of the coenzymes or succinate could “repair” the biochemical lesion. After 24 h of ischaemia the activities of dehydrogenases and diaphorases were markedly decreased. Our data indicate that loss of the reduced coenzymes plays a key role in identifying myocardial infarction with tetrazolium salts. In older infarctions loss of coenzymes is joined by decreased activities of dehydrogenases and diaphorases. The principal mechanisms of staining is an enzymatic cycling.

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  1. Altman FP (1972) Quantitative dehydrogenase histochemistry with special reference to the pentose shunt dehydrogenase. In: Progress in histochemistry and cytochemistry, vol. 4. G. Fischer, Stuttgart, pp 225–271

  2. Bernofski C, Swan M (1973) An improved cycling assay for nicotinamide adenine dinucleotide. Anal Biochem 53:452–458

  3. Brdiczka D, Pette D, Brunner G, Miller F (1968) Kompartimentierte Verteilung von Enzymen in Rattenlebermitochondrien. Eur J Biochem 5:294–304

  4. Bretschneider HJ (1972) Die hämodynamischen Determinanten des myokardialen Sauerstoffverbrauchs. In: Dengler HJ (ed) Die therapeutische Anwendung β-sympathicolytischer Stoffe. Schattauer, Stuttgart, pp 45–60

  5. Burch HB, Bradley ME, Lowry OH (1967) The measurement of triphosphopyridine nucleotide and reduced triphosphopyridine nucleotide and the role of hemoglobin in producing erroneous triphosphopyridine nucleotide values. J Biol Chem 242:4546–4554

  6. Doerr W (1950) Über die Anwendung des Reduktionsindikators Triphenyltetrazoliumchlorid (TTC) in Histologie und Histophysiologie. Frankf Pathol 61:557–573

  7. Flameng W, Winkler B, Wüsten B, Schaper W (1977) Minimum requirements for the measurement of regional myocardial blood flow using tracer microspheres. In: Lewis DH (ed) Recent advances in basic microcirculatory research part I. Karger, Basel, pp 24–29

  8. Gottwik MG, Wüsten B, Sasahara A, Askenazi J (1979) Hämodynamische Effekte von PGE, eine Woche nach experimentellem transfemoralen Verschluß einer Koronararterie. Z Kardiol 68:4, A84

  9. Jennings RB, Reimer KA (1979) Biology of experimental, acute myocardial ischemia and infarction. In: Hearse DJ, de Leiris J (eds) Enzymes in cardiology. John Wiley and Sons, New York, pp 21–57

  10. Jennings RB, Baum JH, Herdson PB (1965) Fine structural changes in myocardial ischemic injury. Arch Pathol 79:135–143

  11. Klein HH (1981a) Enzyme activities in infarcted canine myocardium. In: Schaper W (ed) Experimental myocardial ischemia and infarction. Marcel Dekker, New York

  12. Klein HH, Schaper J, Puschmann S, Nienaber Ch, Kreuzer H, Schaper W (1981b) Loss of canine myocardial nicotinamide adenine dinucleotides determines the transition from reversible to irreversible ischemic damage of myocardial cells. Basic Res Cardiol (accepted for publication)

  13. Klingenberg M (1974) Nicotinamid-adenine-dinucleotide. In: Bergmeyer, HU (ed) Methoden der enzymatischen Analyse. Verlag Chemie, Weinheim, pp 2094–2108

  14. Müller KD, Klein H, Schaper W (1980) Changes of myocardial oxygen consumption 45 minutes after experimental coronary occlusion do not alter infarct size. Cardiovasc Res 14:710–718

  15. Nachlas MM, Shnitka TK (1963) Macroscopic identification of early myocardial infarcts by alteration in dehydrogenase activity. Am J Pathol 42:379–405

  16. Nineham AW (1955) The chemistry of formazans and tetrazolium salts. Chem Rev 55:355–483

  17. Passonneau JV, Lowry OH (1974) Messung durch enzymatic cycling. In: Bergmeyer HU (ed) Methoden der enzymatischen Analyse. Verlag Chemie, Weinheim, pp 2108–2122

  18. Puschmann S, Gottwik M, Klein HH, Regitz V, Schaper W (1980) Experimentelles Infarktmodell: Transfemorale intraluminale LAD Okklusion bei geschlossenem Thorax. Z Kardiol 69:201 (abstract)

  19. Ramkisson RA (1966) Macroscopic identification of early myocardial infarction by dehydrogenase alterations. J Clin Pathol 19:479–481

  20. Sandritter W, Jestädt R (1958) Triphenyltetrazoliumchlorid (TTC) als Reduktionsindikator zur makroskopischen Diagnose des frischen Herzinfarkts. Verh dtsch Ges Pathol 41:165–170

  21. Schaper W, Frenzel H, Hort W (1979) Experimental coronary artery occlusion. I Measurement of infarct size. Basic Res Cardiol 74:46–53

  22. Wenk H, Ritter J, Meyer U (1970) Beitrag zum histochemischen Nachweis pyridinnucleotidabhängiger Dehydrogenasen; der Einfluß von Coenzym und Phenazinmethosulfat auf die histotopochemische Lokalisation. Acta Histochem (Jena) 37:379–396

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Correspondence to H. H. Klein M.D..

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Klein, H.H., Puschmann, S., Schaper, J. et al. The mechanism of the tetrazolium reaction in identifying experimental myocardial infarction. Virchows Arch. A Path. Anat. and Histol. 393, 287–297 (1981).

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Key words

  • Myocardial infarction
  • Tetrazolium salts
  • NAD
  • Oxidoreductases