Eberhard Seidler

The Tetrazolium-Fomazan System: Design and Histochemistry

Our increasing knowledge about the chemistry and the correlations between chemical structure and histochemical properties of the tetrazolium/formazan system is resulting in: a better understanding of existing histochemical tetrazolium techniques; the selection of optimal tetrazolium salts for qualified use in histochemistry, cytochemistry and biochemistry; both qualitative and quantitative improvements in histochemical techniques for purposes demonstrating the activities of various dehydrogenating enzymes; an extended insight into the state of the tested biological object by means of tetrazolium indicators with special properties; and the combination of histochemical enzyme determination with further morphological techniques. This article has attempted to illustrate the progress in the use of the tetrazolium/formazan-system for histochemical purposes.

Histophotometric evaluation of glutamate dehydrogenase activity of the rat hippocampal formation during postnatal development with special reference to the glutamate transmitter metabolism

Summary1.Transmitter glutamate/aspartate synthesis is known to proceed along different metabolic pathways. In this light, the functional relevance of glutamate dehydrogenase in postnatally maturing glutamatergic/aspartatergic structures was studied by means of quantitative enzyme histochemistry.2.The basic requirements concerning the kinetics and calibration of the histochemical glutamate dehydrogenase reaction used were proved to be met in order to obtain valid quantitative data.3.The histochemically demonstrable activity of glutamate dehydrogenase (EC 1.4.1.3) in the hippocampal formation of the rat increased markedly during postnatal development. On day 30, the distribution pattern observed was similar to that in adult animals.4.While the enzyme activity rose within cell body layers from day 0 to day 30 by 240–285%, the increase in neuropil layers was found to be up to 830%. Maximum values were seen in the stratum lacunosum-moleculare of CA1 and CA3 and the stratum moleculare of the dentate fascia on day 30.5.Since the hippocampal neuropil is supposed to be copiously provided with glutamatergic (and aspartatergic?) structures which become functional in rats during the first weeks of postnatal life, the increase in enzyme activity is discussed to be primarily a consequence of maturing synaptic systems using glutamate and/or aspartate as transmitters.

Die Eignung verschiedener Ditetrazoliumsalze als Reduktionsindikatoren in der Enzymhistochemie

Zusammenfassung Die histochemische Prufung von 15 Ditetrazoliumsalzen an Rattenorganen (Herz, Muskel, Leber, Niere und Gehirn) bei Nachweis der Enzyme Succinodehydrogenase, Lactatdehydrogenase und Diaphorase I ergab, das das NBT weiterhin als das „allround“-Tetrazoliumsalz fur den Dehydrogenasenachweis anzusehen ist. Fur spezielle Fragestellungen ist es zweckmasig, auch andere Tetrazoliumsalze zu verwenden.

Histophotometry--the method of choice in quantifying dehydrogenase histochemistry.

Succinate, malate, and lactate dehydrogenase were demonstrated histochemically and measured histophotometrically in the heart and skeletal muscle (m. extensor digitorum longus and m. soleus) of rats at different ages. To prove the value of histophotometry, the enzymes of the tissues were estimated biochemically. The gel film technique cannot sufficiently prevent the diffusion of the soluble enzymes (malate-, lactate dehydrogenase) out of the tissue sections. Because of the different mobility, various isoenzymes, histophotometry cannot give reliable results. But, as far as membrane-bound dehydrogenases (succinate dehydrogenase) are concerned, histophotometry is the method of choice for basic measurements as in routine practical work, especially with tissues where the enzyme activities are heterogeneously distributed, e.g. in different types of muscle fibres in skeletal muscles.

Zur Substantivität von Tetrazoliumsalzen in der Histochemie

SummaryThe known correlations between substantivity and chemical constitution of tetrazolium salts used in histochemistry are further examined on several tetrazolium salts. The investigations are demonstrating that number and position of the nitro groups in the molecule are less important for substantivity of the salt than the influence of a linear, aromatic substitution on N(3) of the tetrazol ring or of doubling the molecule. It is to differentiate between substantivity of tetrazolium salts and the substantivity of the formazans.ZusammenfassungDie in der Literatur beschriebenen Beziehungen zwischen Substantivität und chemischem Bau der in der Histochemie verwendeten Tetrazoliumsalze wurden an weiteren Tetrazoliumsalzen überprüft. Die Untersuchungen ergaben, daß die Anzahl und die Stellung der Nitrogruppen im Molekül an Bedeutung für die Substantivität des Tetrazoliumsalzes hinter dem Einfluß einer linearen, aromatischen Substitution am N(3) des Tetrazolringes bzw. einer Molekülverdoppelung zurücktreten. Es ist zwischen einer Substantivität der Tetrazoliumsalze und einer Substantivität der Formazane zu unterscheiden.

Zur Rolle der Superoxiddismutasen fur den histochemischen Dehydrogenasenachweis

During the incubation of cryostat sections (various organs of rat) with aqueous media xanthine dehydrogenase and superoxide dismutase activities become soluble and detectable in the solution by biochemical methods. Using the tetrazolium technique the demonstration of xanthine dehydrogenase is successful only after separation from the superoxide dismutase by gel electrophoresis. The soluble flavine enzymee xanthine dehydrogenase is taken as an example to discuss the significance of superoxide dismutases in incubation media for the histochemical demonstration of dehydrogenating enzymes by tetrazolium methods.

1-Methoxy-5-methylphenaziniummethylsulfat — ein lichtstabiler Redoxmediator für die Histochemie

Summary 1-Methoxy-5-methylphenazinium methyl sulfate (MPMS) was tested for use as redox mediator in histochemistry of dehydrogenases. Aqueous solutions of MPMS are absolute lightstable. Like PMS or Meldola Blue the succinate dehydrogenase reaction was increased by MPMS. In an nonenzymatic NADH-NBT-test the reaction rate of MPMS exceeds the rates of PMS or Meldola Blue.

Nichtenzymatische Modelle der histochemischen Tetrazoliumreduktion

Zusammenfassung 4 nichtenzymatische biochemische Systeme (NADH-Mediator-Tetrazoliumsalz; NADHRiboflavin- Tetrazoliumsalz ; Methionin-Riboflayin-Tetrazoliumsalz ; Kaliumsuperoxid-Tetrazoliumsalz) wurden auf ihre Eignung als Modelle der histochemischen Tetrazoliumreduktion geprtilt. Das N ADH-PMS-Tetrazoliumsalz-System erwies sich als besonders geeignet. Als Beispiele fur die Anwendungsmogliehkeiten wurden die Reduktionsgeschwindigkeiten einiger in der Histochemie gebrauchlicher Tetrazoliumsalze, die Erhohung der Reduktionsraten durch die Detergeritien Tween , Triton X-l00 und N-Cetylpyridiniumchlorid sowie die Mediatorwirkung einiger histologischer Farbstoffe (Oxazine, Phenazine, Thiazine) bestimmt. Die vorgelegten Ergebnisse werden im Hinblick auf die Rolle der Detergentien und des Sauerstoffs fur den Mechanismus der Tetrazoliumreduktion diskutiert.

On the mechanism of the multistep reduction of tetrazolium salts with special reference to the involvement of tetrazolium radicals

Thin layer chromatography (TLC) was performed to analyze which products are formed when unstable tetrazolinyl radicals are generated during the reduction of tetrazolium salts under physiological conditions of biochemical and histochemical assays. It was found in aqueous media that irrespective the assay conditions, reduction of 2,3-di(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (p-DNTTC) or 2,3-di(4-nitrophenyl)-5-t-butyl-2H-tetrazolium chloride (t-butyl-DNTC) always lead to production of formazan and never to generation of the corresponding 2(4-nitrophenyl)-5-phenyl- (respectively t-butyl-) tetrazoles by splitting off a nitrophenyl side chain from the tetrazolium ring as a cation. This reaction is known to occur in non-aqueous media (Neugebauer, 1973). Because p-DNTTC is analogous to tetrazolium salts that are used in biochemical and histochemical assays, it is concluded that reduction of tetrazolium salts leads to formation of formazans only under these conditions.

Zum histochemischen Nachweis von Gewebsalterationen durch Tetrazoliumsalze unterschiedlicher Nitrosensitivität

Zusammenfassung Mit dem Nachweis der Succinatdehydrogenase am Myokard der Ratte wird gezeigt, das durch die atypisch-farbigen Formazane geeigneter Nitromonotetrazoliumsalze Zellalterationen (Isoprenalinmodell) unterschiedlich gut zu erfassen sind. Die verschieden starke Neigung der Nitromonotetrazoliumsalze, histochemisch mit ihrer atypischen Formazanform zu reagieren („Nitrosensitivitat”), ist durch ihren chemischen Bau bestimmt. Fur den verbesserten histochemischen Nachweis von Zell- und Gewebsalterationen wird eine Reihe von Nitromonotetrazoliumsalzen mit abgestufter Nitrosensitivitat angegeben.