The red impurity in trypan blue

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<ul><li><p>868 Specialia J~XPERIENTIA 26/8: </p><p>t ions (usually 0.02-0.06 ml) was added to the bath ing medium. Every 10-15 rain, another smal l volume of the test solut ion was added to the bath ing medium to produce a step-wise increase in the concentrat ion of the test compounds, unt i l the heart went into a systolic arrest. </p><p>The results are summar ized in the Table. As can be seen from this table, despite the absence of 3/%hydr0xy group, 3-deoxydigitoxigenin produced almost as strong a cardiotonic act ion as digitoxigenin in this preparat ion. Very recently, ZI~RCHER et al. 5 have reported that 3-deoxydigitoxigenin could induce a marked inhib i t ion of a t ranspore ATPase prepared from the heart muscle of the guinea-pig, a f inding which is in good accordance wi th ours. </p><p>Actions of digitoxigenin and 3-deoxydigitoxigenin on the isolated frog's heart (STRAUB'S preparation) </p><p>Concentration 10 s 3 10 -s 10 -~ 3 x 10 -7 (g/ml) </p><p>/_~ -- + x + + x </p><p>Digitoxigenin + -- x l - - + + x ( - - + + x </p><p>-- + 3-Deoxydigitoxigenin - - + x </p><p>+ + x + + x </p><p>--, no effect; +, improvement of contractility without a tendency to systolic arrest; systolic arrest. </p><p>In a previous communicat ion a, we reported that 14-deoxy-14/3H-uzarigenin retains cardiotonic act iv ity, despite the absence of 14/%OH. Thus, it is now clear that , against the long-standing belief on the structure- act iv i ty relat ionship of the cardiotonic steroids, ne i ther 3/3-OH nor 14~-OH is indispensable for the cardiotonic act ion of the compounds. </p><p>A pre l iminary report of the present s tudy was read a t the 42nd Annua l Meeting of the Japanese Pharmaco- logical Society held in Tokyo on the 2Ild of April, 19696. </p><p>Zusammen/assung. Die I-terzwirksamkeit yon 3-Deoxy- digitoxigenin am isolierten Froschherzen wurde gepriift. Im Gegensatz zur bisherigen Auffassung (3/~-Hydroxy- gruppe notwendig ftir kardiotonische Wi rkung der Digi- ta l is -Verbindungen) zeigte 3-Deoxydigi toxigenin eine starke kardiotonische Akt iv i tgt . </p><p>K. TAKEDA 7, T. SHIGEI 7 and S. IMAIS </p><p>Department o/Pharmacology, Institute/or Cardiovascular Diseases, and Faculty o/ Medicine, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo (Japan), 18 February 1970. </p><p>5 W. ZURCHER, ]~. WEISS-BERG and CH. TAMM, Helv. chim. Acta 52, 2449 (1969). </p><p>6 T. SHIGEI, K. TAKEDA, S. MINESHITA, S. IMAI and M. OKADA, Folia pharm, jap. 65, 29 w (1969), in Japanese. </p><p>7 Department of Pharmacology, Institute for Cardiovascular Dis- eases, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo (Japan). </p><p>s Department of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo (Japan). </p><p>The Red Impur i ty in T rypan Blue </p><p>Dyes such as t rypan blue, Evans blue and Niagara blue 2t3, prepared by coupl ing benzidine or a der ivat ive wi th an aminonaphtho ld isu lphon ic acid, were introduced into biology as v i ta l stains and later used in the measure- ment of blood volume. Publ icat ions on these biological uses conta in numerous references to compl icat ions arising from the invar iable presence of contaminat ing red compounds in samples of the dyes. The first systemat ic study, of the red impur i ty in t rypan blue, was made by KELLY ~, whose paper also includes a summary of the earlier l i terature. Fur ther interest in the red impuri t ies followed the discovery that t rypan blue was both terato- genic ~ and carcinogenic a, and subsequent reports of var iable responses to different commercia l samples of t rypan blue led to suggestions that the red impur i ty might be the act ive principle of the whole dye. Experi - ments on fract ions isolated from commercia l t rypan blue suggest that this is not so: the teratogenic act iv i ty of t rypan blue is probab ly due to its major blue component ~, 5, whereas the carcinogenic act iv i ty appears to reside in a fur ther 'purple' impur i ty present in some samples 6. However, i t is not certain whether the red impur i ty con- t r ibutes to the teratogenic or carcinogenic potency of the whole dye. An invest igat ion of th is quest ion requires more of the compound than can convenient ly be prepared by extract ion from the whole dye. We have therefore determined the structure of the red impurit ies of Niagara </p><p>blue 2B (Colour Index no. 22610) and t rypan blue (Colour Index no. 23850) wi th a view to prepar ing synthet ic samples in quant i ty for metabol ic and toxicological studies. </p><p>A small quant i ty of the red impur i ty of Niagara blue 2]3, extracted from the whole dye as described by BECK and LLOYD ~, was reduced wi th dithionite, acidified and the decolourized solution passed through a column of Dowex 1 (chloride form) to remove anionic mater ials. The eluate gave an absorpt ion spectrum in 0.1N tIC1 and 0.1N NaOH identical w i th that of 4-aminobiphenyU. Paper electrophoresis of the reduct ion products by the method of LLOYD and BECK s was consistent wi th these </p><p>1 j . W. KELLY, Stain Technol. 53, 79 (1958). 2 j . GILLMAN, C. GILBERT, T. GILLMAN and I. SPENCE, S. Aft. J. </p><p>reed. Sci. 73, 47 (1948). a j . GILLMAN, T. GILLMAN and C. GILBERT, S. Afr. J. med. Sci. 74, </p><p>21 (1949). 4 F. BECK and J. B. LLOYD, J. Embryol. exp. Morph. ll, 175 (1963). 5 A. N. BARBER and J. C. GEER, J. Embryol. exp. Morph. 72, 1 </p><p>(1964). 6 j. DIJKSTRA and J. GILLMAN, Nature 79J, 803 (1961). 2 A. R. KATRITZKY and P. SIMMONS, J. chem. Soc. 1960, 4901. a j . B. LLOYD and F. BECK, Stain Technol. 39, 7 (1964). </p></li><li><p>15.8. 1970 Specialia 869 </p><p>being 4-aminobiphenyl and 2, 8-d iamino- l -naphthol -3 ,6- disulphonic acid, indicat ing that the red impur i ty of Niagara blue 2B is 8-amino-2-(4 ' -b iphenylazo)- l -naph- thol -3,6-disulphonic acid (I). This compound was then synthesized, by coupl ing diazotized 4-aminobiphenyl wi th 8-amino- l -naphthol -3 ,6-d isn lphonic acid (H-Acid) in alkal ine solution, and found to be identical in absorpt ion spectrum and chromatographic behav iour w i th the red in :pur i ty of N iagara blue 2B. I t presumably arises as a by-product in the synthesis of Niagara blue 2B, th rough H-Acid coupl ing at one but reducing the second diazonium group of a tetrazot ized benzidine. A paral lel s tudy wi th the red impur i ty of t rypan blue shows that this is (II), the 3, 3 ' -d imethyl analogue of (I) (see formula). Other </p><p>possible structures for the red impur i ty of t rypan blue were considered, such as (II) pars-substituted in the b ipheny l moiety by OH (suggested by WEISEg), NH~ or C1. These could arise by incomplete tetrazot izat ion of benzidine or by incomplete coupling followed by subst i tut ion of the remain ing d iazonium group. Each of these possibil it ies was e l iminated by synthesis of the appropr iate b iphenyls and of the monoazo dyes result ing from their alkal ine coupl ing wi th H-acid :0. </p><p>Zusammenfassung. Isol ierung und StrukturaufklS~rung der roten Komponente des teratogenen und karz inogenen Farbstof fs T rypanb lau : 8-amino-2-(3', 3" -D imethy lb iphe- nyl-4' azo)-naphth- 1-ol- 3, 6-Disulphons~ure. </p><p>CH a CH3 NFI 2 \ / HO \ </p><p>/ SOaNa </p><p>Red Impurity of Trypan Blue (II) </p><p>j . B. LLOYD and F. E. FIELD </p><p>Department o/ Biochemistry, University College, Cardi// (Wales), 23 February 7970. </p><p>2 R. WEtSE, Z. wiss. Mikrosk. 5d, 398 (1937). 10 We thank Tenovus for a grant in aid of this work. </p><p>I nduct ion by K i l led Post -Noda l F ragments of the Def in i t ive P r imi t ive S t reak in the Ch ick ~ </p><p>I t has been well establ ished that the l iving Hensen's node of the chick b lastoderm induces neural differentia- t ion when grafted under competent chick ectoderm (WADDINGTON ~, WOODSlDE 3, GALLERA and CASTRO- CORREIA 4, PASTBt~NAK and McCALLIO~ 5, and VAKAET6). I t has also been clearly establ ished that Hensen 's node retains its induct ive capaci ty after it has been kil led with 70% alcohol or heat (WADDINGTON 2 and LEIKOLA and McCALLIONY). Fur thermore, kil led Hensen's node also exerts an induct ive inf luence on competent amphib ian ectoderm (VlSWA~ATH, LXlKOLA and TOIVONENS). </p><p>The max imum induct ive capacity of the definit ive pr imit ive streak is located in Hensen's node. This qual i ty of the streak diminishes rapidly towards the posterior end of the streak (GALLERA9). I t is well known from a large number of studies that f ragments of the def init ive primi- t ive streak taken more than 0.8 mm behind the node, when implanted into young host embryos, are incor- porated into the host embryo and exert no induct ive inf luence on the epiblast of the host. At best, these tissues only contr ibute to the per ipheral mesoblast of the host (GALLERA9 and W'AHEED and McCALLION :0). I t has been shown, however, that under certa in condit ions of pre- t reatment wi th such substances as cysteine or l i th ium chloride poster ior f ragments of the definit ive pr imit ive streak acquire some induct ive capaci ty (WAt~EED and MULHERKAR 11 and WAHEED and McCALLIONI~ Under these condit ions the implanted f ragment retains its integrity, prol i ferates and dif ferentiates mesoderm and often chorda and exerts some neural iz ing influence on the host ectoderm. </p><p>The present s tudy was under taken in order to discover whether kil led f ragments of the posterior par t of the definit ive streak could exert any induct ive act ion on competent ectoderm. Under these condit ions the frag- ments would reta in the i r integr i ty but would not pro- l i ferate or differentiate. </p><p>The embryos used in these exper iments were obta ined from Whi te Leghorn eggs supplied by a commercia l hatchery. The eggs were incubated for 14-16 h at 39 ~ Small f ragments Of the pr imit ive streak, taken 1.0 mm or more posterior to the node of definit ive pr imit ive streak blastoderms, kil led in 70% alcohol and thoroughly r insed in normal saline solution were used as implants. B lastoderms somewhat younger than the def init ive streak stage were explanted and cultured at 39 ~ according to the method of NEW :2. The b lastoderms were brief ly exposed to UV- i r radiat ion in order to retard their rate of development, and the implants were inserted between the epibl~st and the hypob last at the per iphery of the embryos. The purpose of the i r radiat ion was to prevent the isolation of the imp lant from the epiblast by the outgrowth of lateral mesoderm (LmKoLA and MCCAL- LIONY). The embryos were examined after 24-30h in culture. They were photographed as l ight ly sta ined whole mounts. Subsequently, the embryos were embedded in </p><p>1 This work was supported by a grant from the National Research Council of Canada to D. J. McCALLION. </p><p>2 C. H. WADDIN~TON, J. exp. Biol. 10, 38 (1933). a G. L. WOODSlDE, J. exp. Zool. 75, 259 (1937). 4 j . GALLERA and J. CASTRo-CoRREI*, C.r. Soc. Biol., Paris 75d, </p><p>1728 (1960). 5 L. PASTERNAK and D. J. McCALLION, Can. J. Zool. 40, 585 (1962). 6 L. VAKAET, C. r. Soe. Biol., Paris 159, 232 (1965). 7 A. LEIKOLA and D. J. McCALLION, Can. J. Zool. d6, 205 (1968). 8 J. R. VIS\VANATH, A. LEIKOLA and S. TOIVONEN, Experientia 25, </p><p>38 (1969). 9 j . GALLERA, Bull. Ass. Anat. 725, 632 (1965). 10 M. A. WAHEED and D. J. McCALLION, Ann. Zool. Fenn. 7, 67 </p><p>(1970). :: lVI. A. WAHEED and L. MULHERKAR, J. Embryol. exp. Morph. 77, </p><p>161 (1967). 12 D. A. T. NEw, J. Embryol. exp. Morph. 3, 326 (1955). </p></li></ul>