Triphenyltetrazolium chloride reaction

The well-known triphenyltetrazolium chloride (TTC) reaction for the detection of a-ketolsteroids, pyridinium carbinols and pyridinium glycols can also be included here [20-23]. The chromophore system of the red-colored formazan dye produced by reduction of the TTC is composed of highly conjugated double bonds resulting from the combination of a phenylhydrazone group with an azo group ... 

Tetrazolium salts are unstable in basic solutions yielding intense colors. This reaction is still little understood.233,234 In the reaction of 2,3,5-triphenyltetrazolium with hydroxide, it is postulated that a hydroxide ion is involved first as a counterion later leading to the hypothetical A-hydroxy-formazan (147).229 Weiner studied the kinetics of this reaction and identified 1,3,5-triphenylformazan in 10% yield. In concentrated alkaline solutions, the A-hydroxytetrazole (148) has been isolated from triphenyltetrazolium chloride (Scheme 19).235,236... 

The oxidation of the a-ketol moiety present in the steroid under examination and the subsequent reduction of triphenyltetrazolium chloride to the corresponding triphenylformazan are depicted in the following reaction ... 

The dihydroxyacetone side-chain (554) in Reichstein s S is oxidized by alkaline triphenyltetrazolium chloride to the 21-aldehyde (555) further reaction under the influence of alkali affords a salt of the 17a,20-dihydroxypregnan-21-oic acid (556).22 ... 

Upon selenium dehydrogenation sabine yielded a product with UV-absorption identical with that of cevanthridine (CCVIII-A), a selenium dehydrogenation product of cevine and related alkaloids (109). On this basis it was concluded that sabine possesses the cevane (CCVIII-B) ring system. The failure of sabine to give the characteristic cevine color reaction with sulfuric acid and to form a potassium salt were interpreted to indicate the absence of a masked a-ketol system (109). The latter conclusion was advanced independently on the basis of the failure of the alkamine to isomerize under alkaline hydrolytic conditions or to reduce triphenyltetrazolium chloride reagent (63). 

Reduction Tests. Corroborative data pertaining to further identification of these isolated spots can be obtained by subjecting the strips containing the steroids to reduction tests, such as the alkaline silver nitrate, triphenyltetrazolium chloride (2), or potassium permanganate color reactions, or to other types of characterizing procedures, which take advantage of the molecular structures of the compounds under consideration. 

The sequence of reactions from 3 to 7, Fig. 5, was established in the following manner. Dehydrogenation of isobutyryl CoA was demonstrated with dialyzed liver extract, employing the oxidation-reduction indicator, triphenyltetrazolium chloride, as the hydrogen acceptor (87). Only the CoA derivate would serve as a substrate. Furthermore, Crane et al. (97) observed that the flavoprotein, butyryl CoA dehydrogenase, specific for straight chain acyl CoA derivatives from C4 to Cg, is reduced by isobut3rryl CoA. 

The oxidation of isovaleryl CoA to /8-methylcrotonyl CoA (Fig. 6, reaction 3) is analogous to the corresponding reaction with straight chain fatty acids (see Volume I, Chapter 7). Evidence for the dehydrogenation of isovaleryl CoA was secured by incubating this compound with dialyzed rat liver extract in an incubation mixture containing triphenyltetrazolium chloride (116). The oxidation-reduction indicator was reduced at the rate of 0.25 /xmoles per hour. While it was not feasible to identify the /8-methyl-crotonyl CoA formed because of the inability to separate it from the isovaleryl CoA, further evidence of this reaction was obtained by showing the conversion of the isovaleryl CoA to -hydroxyisovaleryl CoA due to presence of enoyl hydrase (crotonase) in the enzyme preparation. 

Hence, for example, the reaction of triphenyltetrazolium chloride with a ketol in a weakly alkaline medium may be written as... 

An extensive investigation on the polarography of tetrazolium salts was accomplished by Jambor and co-workers [167-173]. They found that at pH > 6.0 triphenyltetrazolium chloride is reduced to triphenylformazane and then to benzhydrazidine (see the Ried and Wilk scheme), and that at pH < 6.0 the wave corresponds to two reactions ... 

L-Lysine is converted by an enzyme from a strain of Pseudomonas into y-aminovalerianic acid, NH3, and COg. The rate of incorporation of in dry matter is fairly important. These results point to the frequent occurrence of reactions such as (91) and many others similar and unknown. In the L-lysine oxidase experiments oxygen consumption cannot be affected by crystalline catalase. Methylene blue, triphenyltetrazolium chloride, and 2,6-dichlorophenolindolephenol cannot replace oxygen. 

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