The reaction of ethylene oxide with some proteins, amino acids and vitamins
A report that the ethylene oxide fumigation of animal diets reduced their nutritive value has prompted a more detailed study of the reactions of this commercially-used fumigant. It has been demonstrated that the 24-hour fumigation of commercial casein, egg albumin and lactalbumin with ethylene oxide greatly reduced the ability of these proteins to support the growth of weanling rats when the proteins were fed as 9% of a purified diet as the sole source of amino acid nitrogen. Thorough drying of the proteins reduced the extent of subsequent fumigation damage. The nutritional quality of the three proteins was completely restored by the supplementary feeding of L-histidine-HCl, DL-methionine, and L-lysine-HCl, or, in some cases, only one or two of these amino acids. Microbiological assay confirmed a reduction in the biological availability of 11% to 83% of the histidine, 6% to 55% of the methionine and 51 to 92% of the lysine, depending on the protein and the amount of moisture present during fumigation. Histidine was generally the most labile amino acid and lysine the least labile. Additional amino acids appeared to be affected when an hydrolysate of casein was fumigated.
The ethylene oxide fumigation of soybean alpha protein improves its growth-promoting quality for the rat. Evidence is presented that fumigation inactivates the heat-labile proteinaceous growth inhibitor, long recognized as a component of soybean protein.
Chemical justification for the destructive action of ethylene oxide on the biological value of histidine, methionine, and certain B-vitamins was sought by reacting model compounds in aqueous solution with the fumigant and identifying the products. At 25°C. and atmospheric pressure ethylene oxide readily hydroxyethylates tertiary as well as primary and secondary amine nitrogen and also the sulfur of thioethers. Imidazole and histidine yield the 1,3-bis-(2-hydroxyethyl)imidazolium derivatives; nicotinamide, nicotinic acid, and pyridine yield the corresponding N-(2-hydroxyethyl)pyridinium compounds; and N-acetylmethionine is converted to s-(2-hydroxyethyl)-N-acetylmethionine, thetin. The reaction of ethylene oxide with amino acids results in hydroxyethylation of the primary amino groups but no esterification of carboxyl groups was observed. The extent of ethylene oxide-imidazole reaction in fumigated proteins could be determined colorimetrically.
In the presence of ethylene oxide the mercapto group of cysteine appears to become doubly alkylated to produce a sulfonium compound which causes a flaccid paralysis, respiratory failure and death when injected subcutaneously into weanling rats. The structure of the compound bears certain similarities to other synthetic spasmolytics. Evidence is presented that the reaction of ethylene oxide with pyridoxine, riboflavin and folic acid involves the hydroxyethylation of tertiary heterocyclic nitrogens. A striking resemblance was observed between the alkylating capacity of ethylene oxide and the sulfur and nitrogen mustards.
New compounds described are 1,3-bis-(2-hydroxyethyl)imidazolium chloride; N¹-(2-hydroxyethyl)nicotinic acid, betaine; and N-(2-hydroxyethyl) nicotinic acid chloride. N-(2-hydroxyethyl)nicotinamide chloride was also crystallized and its melting point found to be 14 degrees centigrade higher than previously reported in the literature. Other reaction products were studied in solution or as solid adducts of reinecke's salt or phosphotungstic acid.
The proteolytic activity of chymotrypsin was progressively reduced by exposure of the cystalline enzyme to ethylene oxide vapor or by treatment of the enzyme in aqueous solution with the fumigant. However, treatment of chymotrypsin in aqueous solution with ethylene oxide did not impair its ability to hydrolyze p-nitrophenyl acetate. Likewise, the imidazole groups of native chymotrypsin appear to be protected from the hydroxyethylating action of ethylene oxide. Denaturing the protein in 8 M urea uncovered the imidazole groups and permitted reaction with the fumigant.
On the basis of these studies it is felt that the effect of existing commercial ethylene oxide fumigating processes on the nutritive value of foods and feedstuffs bears closer investigation.