- Email: [email protected]
242. Effect of colourings on the haemoglobin-nitrite reaction
PRESERVATIVES
279
241. The effect of nitrite on erythrocytes Previous work has shown that nitrite (I) oxidizes haemoglobin and reduced glutathione in the red blood cell to methaemoglobin and oxidised glutathione respeCtively (Keilin & Hartree, Nature, Lond. 1946, 157, 210; Vella, Experientia 1959, 15, 433). Both of these studies however employed cells that had been deprived of glucose or other sources of energy. The present paper reports the results of an investigation into the influence of the cell's sources of energy on the depletion by I of reduced glutathione. It was found that glucose prevented high concentrations of I oxidising glutathione, but did not affect the oxidation of haemoglobin, in the absence of glucose, oxidation of glutathione took place. Lactate however failed to prevent either the oxidation of glutathione or haemoglobin. Also in erythrocytes taken from rats that lacked an enzyme essential to normal pentose sugar metabolism, oxidation of glutathione followed their exposure to I in contrast to normal erythrocytes.
The results suggest that in vivo, I oxidised haemoglobin in preference to glutathione which is protected from oxidation by the processes involved in normal pentose sugar metabolism. The buffering effect that haemoglobin exerts in this respect allows glutathione to maintain its role in protecting the erythrocyte from spontaneous rupture, enzyme inactivation and other damage, manifest by the production of Heinz bodies. Harley, J. D. & Robin, Helen (1962). The effect of the nitrite ion on intact human erythrocytes. Blood 20, 710. 242. Effect of colourings on the haemoglobin-nitrite reaction The mode of action of Trypan Blue and Evans Blue in producing teratog'enic effects (Cited in F.C.T. 1963, 1, 152) is as yet unknown. Foetal abnormalities of course also result in certain dietary deficiency states of which an example is riboflavine (I) deficiency. In this connection it is of interest that the resistance of intracellular haemoglobin (II) to oxidation by nitrite ion is known to be influenced by changes in the state of I metabolism (Metcalf, Phys. Med. Biol. 1961, 6, 427). These facts prompted the present investigation into the effect of Trypan Blue and Evans Blue on the reaction between II and sodium nitrite (III) to produce methaemoglobin (IV). It was found that" Trypan Blue and Evans Blue accelerated by up to 50 % the rate of conversion of II to IV. It was suggested that II became more sensitive to oxidation by III as a result of the formation by these colourings of stable compounds with the protein component of flavoprotein enzymes (in the red blood cell II is normally prevented from undergoing oxidation to IV by the action of a reduced coenzyme, NADH2, supplies of which might be expected to fail if normal cellular respiration was interfered with by Trypan Blue in the manner suggested). It was shown that under certain circumstances the administration to animals that had been injected with Trypan Blue or I could raise the resistance of II to oxidation by III. The author suggested that the effect of these colourings on the stability of II was linked with their ability to give rise to foetal abnormalities. In support of this view none of the compounds known to be non-teratogenic in the rat including Chlorazole Black, Congo Red, Indian ink, ca rboxymethylcellulose and H-acid were shown to affect the resistance of II to oxidation by III. Metcalf, W. K. (1962). Effect of teratogenic and unrelated dyes on the haemoglobin nitrite sensitivity reaction. Brit. J. PharmacoL 19, 492. '243. Benzpyrene in curing smoke The carcinogenic hydrocarbon, 3,4-benzpyrene (BP) has been identified in smoked meat and fish products (Cited in F.C.T. 1963, 1, 118). The influence of the method of smoke generation on the formation of BP was studied by burning the wood in a reduced (I),
279
241. The effect of nitrite on erythrocytes Previous work has shown that nitrite (I) oxidizes haemoglobin and reduced glutathione in the red blood cell to methaemoglobin and oxidised glutathione respeCtively (Keilin & Hartree, Nature, Lond. 1946, 157, 210; Vella, Experientia 1959, 15, 433). Both of these studies however employed cells that had been deprived of glucose or other sources of energy. The present paper reports the results of an investigation into the influence of the cell's sources of energy on the depletion by I of reduced glutathione. It was found that glucose prevented high concentrations of I oxidising glutathione, but did not affect the oxidation of haemoglobin, in the absence of glucose, oxidation of glutathione took place. Lactate however failed to prevent either the oxidation of glutathione or haemoglobin. Also in erythrocytes taken from rats that lacked an enzyme essential to normal pentose sugar metabolism, oxidation of glutathione followed their exposure to I in contrast to normal erythrocytes.
The results suggest that in vivo, I oxidised haemoglobin in preference to glutathione which is protected from oxidation by the processes involved in normal pentose sugar metabolism. The buffering effect that haemoglobin exerts in this respect allows glutathione to maintain its role in protecting the erythrocyte from spontaneous rupture, enzyme inactivation and other damage, manifest by the production of Heinz bodies. Harley, J. D. & Robin, Helen (1962). The effect of the nitrite ion on intact human erythrocytes. Blood 20, 710. 242. Effect of colourings on the haemoglobin-nitrite reaction The mode of action of Trypan Blue and Evans Blue in producing teratog'enic effects (Cited in F.C.T. 1963, 1, 152) is as yet unknown. Foetal abnormalities of course also result in certain dietary deficiency states of which an example is riboflavine (I) deficiency. In this connection it is of interest that the resistance of intracellular haemoglobin (II) to oxidation by nitrite ion is known to be influenced by changes in the state of I metabolism (Metcalf, Phys. Med. Biol. 1961, 6, 427). These facts prompted the present investigation into the effect of Trypan Blue and Evans Blue on the reaction between II and sodium nitrite (III) to produce methaemoglobin (IV). It was found that" Trypan Blue and Evans Blue accelerated by up to 50 % the rate of conversion of II to IV. It was suggested that II became more sensitive to oxidation by III as a result of the formation by these colourings of stable compounds with the protein component of flavoprotein enzymes (in the red blood cell II is normally prevented from undergoing oxidation to IV by the action of a reduced coenzyme, NADH2, supplies of which might be expected to fail if normal cellular respiration was interfered with by Trypan Blue in the manner suggested). It was shown that under certain circumstances the administration to animals that had been injected with Trypan Blue or I could raise the resistance of II to oxidation by III. The author suggested that the effect of these colourings on the stability of II was linked with their ability to give rise to foetal abnormalities. In support of this view none of the compounds known to be non-teratogenic in the rat including Chlorazole Black, Congo Red, Indian ink, ca rboxymethylcellulose and H-acid were shown to affect the resistance of II to oxidation by III. Metcalf, W. K. (1962). Effect of teratogenic and unrelated dyes on the haemoglobin nitrite sensitivity reaction. Brit. J. PharmacoL 19, 492. '243. Benzpyrene in curing smoke The carcinogenic hydrocarbon, 3,4-benzpyrene (BP) has been identified in smoked meat and fish products (Cited in F.C.T. 1963, 1, 118). The influence of the method of smoke generation on the formation of BP was studied by burning the wood in a reduced (I),