Enzymic reduction of azo food colourings

Fd Cosmet. ToxicoL Vol. 5, pp. 533-534. Pergamon Press 1967. Printed m Great Bntain

SHORT PAPER

Enzymic Reduction of Azo Food Colourings J. W. DANIEL Imperial Chemical Industries Limited, Industrial Hygiene Research Laboratories, Alderley Park, Cheshire, England (Received 3 April 1967)

Introduction Azo compounds are reduced to their component amines by an enzyme, or more probably a group of enzymes, located predominantly in the liver. Despite the fact that previous studies both in the rabbit (Fouts, Kamm & Brodie, 1957) and rat (Manchon, Gradnauer & Lowy, 1964) showed the system to possess low substrate specificity, Jones, Ryan & Wright (1964) reported that the water-soluble, azopyrazolone food colouring tartrazine (C.I. (1956) No. 19140) was not reduced when it was incubated with fortified homogenates of rat liver. To explain this observation Jones et aL (1964) suggested that azopyrazolones are not true azo compounds but that they exist essentially as ketohydrazones stabilized by strong internal hydrogen bonding (Jones, Ryan, Sternhell & Wright, 1963). The subcellular distribution of azo-reductase activity in rat liver fs currently under investigation in this department, and this report presents preliminary results on the rate of reduction of the three water-soluble azo compounds, tartrazine, Orange G (C.I. (1956) No. 16230) and Orange II (C.I. (1956) No. 15510), by the 10,000 × g supernatant of homogenates of male and female rat liver.

Experimental Materials. Tartrazine, Orange II and Orange G were obtained from Imperial Chemical Industries Ltd. (Dyestuffs Division, Blackley, Manchester) and contained not more than 15% of inorganic diluent. The colourings were chromatographically homogeneous and were free of unreacted amines (Daniel, 1962). They were dissolved in water to give a concentration of 10/~moles/ml. NADP, glucose 6-phosphate and glucose 6-phosphate dehydrogenase were obtained from the Boehringer Corporation (London) Ltd. N'-diethyl-N-l-naphthylpropylenediamine hydrochloride was obtained from "Prolabo" of Rh6ne-Poulenc. Preparation o f homogenates. The animals used were from the Alderley Park strain of SPF albino Wistar rats and had a body weight of 220-250 g. Rats were killed by a blow on the neck, the liver removed and homogenized in four volumes of chilled 1.15 % KCI solution using a teflon homogenizer. The homogenate was centrifuged at 10,000 × g for 10 rain in a refrigerated centrifuge (MSE '17') and the supernatant retained. Incubation procedure. Supernatant (2.5 ml), equivalent to 0.625 g liver, was added to 2.5 m.l of a solution containing NADP (1.5/~moles), MgCI 2 (25/zmoles), glucose 6-phosphate (50/zmoles), nicotinamide (100/~moles) and potassium phosphate buffer (280/~moles; pH 7.4) CKato & Gillette, 1965). Glucose 6-phosphate dehydrogenase (0.5 unit) and the substrate (1 ml) were added and the mixture, total volume 6.1 ml, incubated in a metabolic

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shaker for 30 m m at 37 ° with nitrogen or air as the gas phase. The reaction was terminated by the addition o f 0.5 ml conc. HC1. Protein was removed by centrifugation and the supernatant separated and diluted to 10 ml with water. A portion of this solution, usually 2 ml, was taken for the colorimetric analysis o f sulphanilic acid (tartrazine and Orange II) and aniline (Orange G) by diazotization and coupling with N'-diethyl-N-l-naphthylpropylenediamine (Daniel, 1961). Results and Discussion The a m o u n t o f each individual substrate reduced when incubated anaerobically with homogenates o f rat liver are shown in Table I. K a t o & Gillette (1965) observed that liver microsomes f r o m male rats reduced the drug, neoprontosil, at a rate twice that o f microsomes from female rats. Although Orange G is reduced somewhat more rapidly by male than by female rats the difference is probably not significant ( P ( 0 . 0 5 ) and there is no sex difference in the rate of reduction of either tartrazine or Orange II. Table 1

Anaerobicreduction of Orange G, Orange H and tartrazine by rat-liver homogenates

m#moles colour reduced/g liver/30 mm Substrate Orange II Tartrazlne Orange G

Males 7804- 33 (5) 3104- 37 (6) 10564-162 (4)

Females 6864-118 (5) 2744- 29 (4) 792 4-120 (4)

Figures m parentheses refer to the number of experiments and the results are expressed as the mean 4- the standard devlat~on. The reduction of tartrazine was completely inhibited by oxygen and that o f Orange II and Orange G by about 80~o. This effect o f oxygen may, in part, be due to the N A D P H oxidase activity o f the preparation. It is apparent that tartrazine is reduced more slowly than either o f the two other substrates, a fact which m a y be attributed to the preferred configuration suggested for azopyrazolones. It is impossible to afford any explanation for the reported failure to observe reduction o f tartrazine when incubated under, presumably, similar experimental conditions. Acknowledgement--Technical assistance in this investigation was provided by Miss C. D. Hiles.

REFERENCES Daniel, J. W. (1961). The determination of aromatic amino-compounds. Analyst, Lond. 86, 640. Daniel, J. W. (1962). The excretion and metabolism of edible food-colours. Toxic. appl. Pharmac. 4, 572. Fouts, J. R., Kamm, J. J. & Brodie, B. B. (1957). Enzymatic reduction of prontosd and other azo-dyes. J. Pharmac. exp. Ther. 120, 291. Jones, R., Ryan, A. J., Sternhell, S. & Wright, S. E. (1963). The structures of some 5-pyrazolones and derived 4-arylazo-5-pyrazolones. Tetrahedron 19, 1497. Jones, R., Ryan, A. J. & Wright, S. E. (1964). The metabolism and excretion of tartrazme in the rat, rabbit and man. Fd Cosmet. Toxicol. 2, 447. Kato, R. & Gdlette, J. R. (1965). Effect of starvation on NADPH-dependent enzymes in liver microsomes of male and female rats. J. Pharmac. exp. Ther. 150, 279. Manchon, Ph., Gradnauer, S. & Lowy, R. (1964). Etudes sur ractivit6 azor6ductaslque des surnageants d'homog6nat de foie de rat. I. Influence de divers effecteurs. Fd Cosmet. Toxtcol. 2, 349