The ninhydrin and alloxan reactions have been criticized as being nonspecific and unreliable for histochemical studies due to the diffusion, solubility, and fading of their reaction products. This paper presents some modifications of the techniques that appear to render the reactions more reliable for the identification of proteins and peptides possessing terminal α-amino acids. The resulting color complexes are also stable

Azzam, Nabil A.

Shanklin, William M.

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26 Application of the Ninhydrin and Alloxa n Reactions to Tissue Sections* NABIL A. AZZAM AND WILLIAM M. SHANKLIN Department of Anatomy, Medical College of Virginia, Richmond The ninhydrin and alloxan re-actions have been criticized as be-ing nonspecific and unreliable for histochemical studies due to the diffusion, solubility, and fading of their reaction products. This paper presents some modifications of the techniques that appear to render the reactions more reliable for the iden-tification of proteins and peptides possessing terminal a-amino acids. The resulting color complexes are also stable. In 1862 Strecker demonstrated that a mixture of alloxan and a-amino acid in aqueous solution reacted with the generation of carbon dioxide and the concomitant development of a deep blue color which could be at-tributed to the formation of murexide (acid ammonium purpurate). The lat-ter product was isolated as purple-red crystals from the reaction mixture which, in addition, yielded an alde-hyde containing one less carbon atom than the parent amino acid. Hence an oxidative deamination of the amino acid was instigated by the alloxan reagent (Greenstein and Winitz, 1961). The cytochemical application of the alloxan reaction to tissue sections was criticized by Romieu (1925) on grounds of lack of specificity, and by Giroud (1929) because of color diffusi-bility. Serra (1946) quotes Winterstein (1933) as saying that the test was insensitive with fixed material. Ver-cauteren (1951) maintained that the reaction product behaved as an ani-* Supported by U. S. Public Health Service research grant NB-03901 from the National Institute of Neurological Diseases and Blindness. onic dye that stained tissue compo-nents having an affinity for such dyes. Ruhemann (1910, 1911) observed that other reagents may behave as oxi-dizing agents toward amino acids leaving similarly colored end products. Ninhydrin (triketohydrindene hydrate) reacts with a-amino acids in neutral aqueous solution, resulting in the evo-lution of carbon dioxide, the formation of an aldehyde with one less carbon atom than the parent amino acid, and the development of a deep blue col-oration. This is due to the formation of a colored complex, the ammonium salt of diketohydrindylidene diketohy-drindamine (Greenstein and Winitz, 1961). The ninhydrin reaction, as em-ployed in biochemical studies, was applied to tissue sections by Berg (1922). Later, both this reaction and the alloxan reaction were applied to fixed tissue sections by Serra (1946). Using formalin-fixed material, Serra was able to demonstrate a weak al-loxan reaction, and further showed that the ninhydrin reaction, which could only be produced with pro-longed excessive heat, faded rapidly. Vercauteren (1951) indicated that the reaction products of ninhydrin were, to some extent, soluble in water, and thus may be absorbed on cellular structures which are not the site of the reaction. Hutton (1953) was able to stabilize the reaction product for about a week in unerupted molars of hamsters, fixed only by their inciden-tal embedding in celloidin. Sections sprayed with a 0.25% ninhydrin solu-tion in n-butanol required three to four days incubation at 55 C to de-velop maximum color. Notovny and Owens (1960) pre-vented diffusion of amino acids, and their ninhydrin reaction products, from original sites by fixing and de-hydrating the tissues in anhydrous, N. A. AZZAM AND W. M. SHANKLIN peroxide-free dioxane. The color pro-duced by a-amino acids faded within 24 hours, while that produced by protein was stable for seven days or longer. Vainer and Bona (1963) used various fixatives, such as Carnoy and methyl alcohol, and different concen-trations of ninhydrin varying from 0.1 % to 0.4% to study its reaction on peripheral blood. Their reactions were carried out at 80 C for 5 to 30 min-utes, and the staining remained intact for "a long time." Yasuma and Ichikawa (1953) stained the aldehyde groups by the Schiff reagent in an oxidative de-amination reaction brought about by ninhydrin. Alloxan was also used as an oxidative deamination reagent. A serious objection to such use of the Schiff reagent is its tendency to stain other tissue components (direct Schiff reaction), thus confusing the recogni-tion of true protein sites in the sec-tions. Both the ninhydrin and the nin-hydrin-Schiff histochemical reactions have been criticized. Recently, Pucht-ler and Sweat (1962), reviewed the literature and concluded that the nin-hydrin-Schiff method was unreliable for demonstrating proteins. Kasten (1962) took issue with this conclusion and stated that, in the ninhydrin reac-tion, as distinct from the ninhydrin-Schiff reaction, ammonia reacts with reduced ninhydrin (hydrindantin) to form a violet product which is diffusi-ble, unstable, and offers little possi-bility for histochemical application. He observed that acetylating and de-aminating agents block the ninhydrin and alloxan reactions by masking the carboxyl or the amino groups of the a-amino acids. This indicates that the action of ninhydrin in tissues is at the amino group, and qualifies the ninhydrin reaction as an histochemical test for proteins and polypeptides with free and reactive amino groups. Materials and Methods Pieces of rat kidney, liver, small intestine, skeletal muscle, and car-diac muscle were removed after decapitation. Different concentra-tions of aqueous and alcoholic solu-tions of trichloroacetic, phospho-tungstic, phosphomolybdic, and acetic acids were used as fixatives for 24 hours at room temperature. Tissues fixed in aqueous solutions were dehydrated in a series of al-cohols, cleared in xylene, and embedded in Paraplast. Tissues fixed in 80% alcoholic solutions were dehydrated in 95% and ab-solute alcohol, cleared in xylene, and embedded in Paraplast. Both aqueous and alcoholic solu-tions of phosphomolybdic and phos-photungstic acids proved to be poor penetrants and their use was dis-continued. Sections of tissues fixed in 5 % acetic acid in 80% alcohol were treated with ninhydrin, but, only a weak color developed. The follow-ing detailed procedure was fol-lowed: Tissues were fixed in 5% trichloroacetic acid in 80% alcohol for 24 hours at room temperature. They were then transferred to 95 % alcohol and absolute alcohol for one hour each, and after clearing in xy-lene, were embedded in Paraplast. Paraffin sections were mounted on slides with starch paste, instead of egg albumin, to avoid false localiza-tion. Deparaffinization of sections in xylene was followed by going through two changes of absolute al-cohol. Sections were then treated in 1 % ninhydrin in absolute alcohol at 37 C from one to three hours. Over-treatment tended to weaken the re-action. After rinsing in two changes of absolute alcohol, and clearing in xylene, the sections were mounted in Permount. Both 0.1 % and 0.5 % stannous chloride were added to the ninhydrin solution. Pepsin and trypsin digestion pro-cedures were used as well as acety-lation and deamination blocking re-actions (Pearse, 1961). Alternate sections were treated with 1 % al-loxan in absolute alcohol instead of ninhydrin in the above procedures. Comparable sections of the kid-ney, liver, muscle, and intestine were stained by the ninhydrin-Schiff and alloxan-Schiff methods for protein-bound amino groups (Yasuma and Ichikawa, 1953), the chloramine-T Schiff method for pro-tein-bound amino groups (Burstone, 1955), and the acid solochrome cya-nine method for basic proteins (Pearse, 1961). 27 NINHYDRIN AND ALLOXAN REACTIONS Photomicrographs of rat tissue sections fixed in 5% trichloroacetic acid in 80% alcohol and treated with alcoholic ninhydrin or alloxan. Fig. 1-Kidney tubules following al-loxan treatment (X 700). Fig. 4-Cardiac muscle colored by the ninhydrin reaction (X 700). 28 Fig. 2-Liver cells following the nin-hydrin reaction (X 700). Fig. 5-Striated muscle colored by the alloxan reaction (X 700). Fig. 3-Smooth muscle fibers in the small intestine following the alloxan reaction. Arrow points to darkly col-ored submucosa (X 700). Fig. 6-Striated muscle demonstrating ninhydrin-reaction (X 1400). Results and Discussion We observed an intense bluish-violet color in trichloroacetic acid-fixed sections after the ninhydrin treatment. Alloxan stained the tis-sues a rich red color. Both nin-hydrin and alloxan produced intense coloration of proximal renal tubular epithelium and basement mem-branes (fig. 1). Some parenchymal liver cells (fig. 2) were well-stained while others in the same area failed to bind the color complex. Smooth muscle in the small intestine (fig. 3) stained strongly in contrast to the epithelium. The myofibrils and striations in both cardiac (fig. 4) and skeletal muscles (fig. 5 and 6) were intensely stained. The connec-tive tissue fibers of the muscle, liver, and kidney were poorly stained, except in the submucosa of the small intestine where they were intensely stained. Pepsin and trypsin digestion, prior to ninhydrin and alloxan treatment, completely blocked the reaction by digesting the proteins. Both acetylation and deamination blocked the ninhydrin and alloxan reactions and prevented color de-velopment, thus establishing that the ninhydrin and alloxan act on the terminal amino acids of the protein and peptides chains. According to Moore and Stein (1948), the addition of stannous chloride to the ninhydrin solution reduced for ninhydrin, thus inten-sifying the reaction; however, we found that the addition of 0.1 % or 0.5% stannous chloride completely abolished the reaction. The localization and color ob-tained by the ninhydrin reaction or the alloxan reaction were superior to those obtained by the ninhydrin-Schiff or the alloxan-Schiff reac-tions. The chloramine-T Schiff method, where chloramine-T acts as a di-carbonyl compound like alloxan and ninhydrin, stained the tissues with the same intensity and localization as those of ninhydrin-Schiff and alloxan-Schiff, but was less intense N. A. AZZAM AND W. M. SHANKLIN than that of alloxan or ninhydrin alone. The acid solochrome cyanine method stained the tissues rather weakly. Summary The ninhydrin (triketohydrindene hydrate) reaction, used extensively in biochemical work, has not been successfully applied cytochemically, due to lack of specificity, fading, and diffusion artifacts. These faults are effectively overcome by avoid-ing aqueous media and by fixing the tissues in a 5% solution of tri-chloroacetic acid in 80% alcohol, a good precipitant for proteins pos-sessing free NH2 groups. A fixation of 24 hours is followed by custom-ary embedding in paraffin, and de-paraffinized sections are passed through two changes of absolute alcohol, then treated in 1 % nin-hydrin in absolute alcohol for one to three hours at 37 C. The presence of free NH2 groups in the peptide and protein chains produces a blue-violet color. Treated sections re-tained their color for up to six months. Alternate sections were used for the alloxan procedure; the resulting red color showed the same reaction sites. References BERG. W. Ueber Anwendung der Nin-hydrinreaktion auf mikrosko-pische Prapiirate zum Nachweis niederer Eiweisskorper. Pfiuegers Arch. Ges. Physiol. 195: 543-554, 1922. BURSTONE, M. s. An evaluation of histochemical methods for protein groups. J. Histochem. Cytochem. 3: 32-49, 1955. GIROUD, A. Recherches sur la nature chimique du chondriome. Proto-plasma. 7: 72-98, 1929. GREENSTEIN, J. P. AND M. WINITZ. Chemistry of the Amino Acids. New York: John Wiley and Sons, 1961, vol. 2, p. 1301. HUTTON, W. E. Ninhydrin staining of tissue sections. Stain Technol. 28: 173-175, 1953. KASTEN, F. H. Some comments on a recent criticism of the ninhydrin-Schiff reaction. J. Histochem . Cytochem. 10: 769-770, 1962. MooRE, S. AND W. H. STEIN. Photo-metric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem. 176: 367-388, 1948. NOTOVNY, H. M. AND R. G. OWENS. Localization of amino acids for histochemical estimation. Nature 186: 1057-1058, 1960. PEARSE, A. G. E. Histochemistry, The-oretical and Applied. 2nd edition. London: J. and A. Churchill, 1961. PUCHTLER, H. AND F. SWEAT. Some comments on the ninhydrin-Schiff reaction. J. Histochem. Cytochem. 10: 365, 1962. ROMIEU, M. Bull. Histol. Tech . Micro. 92: 185, 1925 (cited from Pearse, 1960, p. 135). RUHEMANN, s. Triketohydrindene hy-drate. J. Chem. Soc. 97: 2025-2031, 1910. RUHEMANN, S. Triketohydrindene hy-drate. V. The analogs of uramil and purpuric acid. J. Chem. Soc. 99: 1475-1477, 1911. SERRA, J. A. Histochemical tests for proteins and amino acids; the characterization of basic proteins. Stain Technol. 21: 5-18, 1946. STRECKER, A. Notiz iiber eine eigen-thumliche Oxydation durch Al-loxan. Leibig's Ann. 23: 363-365, 1862. VAINER, H. AND C. BONA. Reaction a la ninhydrine modifiee pour l'etude histochimique des pro-teines. I. Application a l'etude cytochimique du sang peripheri-que. Acta Histochem. 15: 125-134, 1963. VERCAUTEREN, R. Possible artefacts introduced by the ninhydrin and alloxan reactions in histochemical applications. Nature 167: 819-820, 1951. WINTERSTEIN, A. In Klein's Hand-buch der Pfianzeanalyze. Vienna: Springer, 1933 (cited from Pearse, 1960, p. 136). YASUMA, A. AND T. lcHIKAWA. Nin-hydrin-Schiff and alloxan-Schiff staining. A new histochemical staining method for protein. J. Lab. Clin. Med. 41 : 296-299, 1953. 29 

Application of the Ninhydrin and Alloxan Reactions to Tissue Sections

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Application of the Ninhydrin and Alloxan Reactions to Tissue Sections

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