Many workers, the author among them, have noticed that rats fed on a diet presumably free from vitamine A, grew for a considerable time before decline set in. This is in striking contrast to the rapid decline that sets in when vitamine B is absent. Such anomalies have led to the view that the complete removal of vitamine A from foods is a far more difficult undertaking than was at one time suspected. Drs. Osborne and Mendel record an experiment wherein the diet, consisting of casein, corn starch, dried brewery-yeast and lard, was first purified by boiling three times with absolute alcohol (under a reflux condenser) for one hour - a process which should get rid of every trace of vitamine A - and then offered to rats; the animals grew for some time, despite this purification of the diet.

Dr. Drummond, the English investigator, points out similar difficulties. Until recently he purified his casein by heating for 24 hours in shallow dishes at 102 degrees centigrade, and then subjecting the material to prolonged and continuous extraction with alcohol and ether. Lately, as a result of experiments on the effect of oxidation on vitamines (see next section) he has modified this procedure, whereby the casein is heated as before, but the temperature is allowed to reach 105 degrees centigrade, kept so for 24 to 48 hours, and the material thoroughly stirred from time to time, so that every opportunity for the oxidation of the vitamine may be given. He finds that rice starch, even in the crude form, is almost entirely devoid of vitamine A. As a source of fat, Dr. Drummond suggests cottonseed oil. He finds that orange juice and salt mixture, and probably the yeast extract, are devoid of vitamine A. His "synthetic diet," containing all the necessary factors except vitamine A, is made up as follows: purified caseinogen (protein) 18 parts, purified rice starch (carbohydrate) 52, refined hydrogenated vegetable oil (fat) 15, yeast extract (vitamine B) 5, orange juice (vitamine C) 5, and salt mixture (mineral matter) 5. (References: T. B. Osborne and L. B. Mendel, Journal of Biological Chemistry, volume 45, page 277, 1921; J. C. Drummond and K. H. Coward, Biochemical Journal, volume 14, page 661, 1920).

Effect Of Heat And Aeration Upon Vitamine A

(see pages 167, 208, 209). Perhaps the most notable advance in the study of vitamines has been to show that exposure to air and aeration ("oxidation") is an even more important factor in the destruction of vitamines than is heat, though the latter, of course, plays an important part. This question will again be referred to.

Not so very long ago Dr. Steenbock in this country and Dr. Drummond in England were of the opinion that vitamine A is readily destroyed by heat. Drs. Osborne and Mendel, on the other hand, confirmed earlier observations which led to the belief that this vitamine is quite resistive to heat, or thermo-stabile. Prof. Hopkins of Cambridge has since confirmed Drs. Osborne and Mendel's opinion, but in addition he has shown that vitamine A is rapidly destroyed by exposure to atmospheric oxygen at temperatures ranging from 15 degrees to 120 degrees centigrade. Here is a typical experiment: Two carefully balanced sets of rats were taken, with ten animals in each set. One set received the standard diet with butter previously heated to 120 degrees centigrade in the autoclave for four hours; the other set had the same diet with the butter heated at the same temperature and for the same time, but with simultaneous aeration. (The diet consisted of highly purified caseinogen, potato starch, sucrose, butter and salt mixture. See footnote to page 109, and also page 222).

The aeration was carried out in a flask immersed in an oil bath. By means of a filter pump a stream of air was drawn through the melted fat at the rate of four to five bubbles per second. The rats fed on autoclaved butter showed vigorous growth for a period of 70 days, when the experiment was discontinued. No xerophalmia developed. Those fed on aerated butter showed a slight growth at first, but then they declined. Three of these rats showed xerophalmia symptoms on the 40 day, three on the 42, three on the 53, and one on the 64 day. "Twelve hours' exposure to 120 degrees centigrade (considerably above the boiling point of water) seems undoubtedly to involve some destruction of vitamine A, but in the absence of air it is far from being completely destroyed. Butter exposed in thin layers at 15 to 25 degrees Centigrade (59 to 77 degrees Fahrenheit) for periods of about a week was found to have lost its power of maintaining the growth or health of rats." As already indicated, the oxidation method probably suggests the best means for ridding experimental diets of any vitamine A they may contain.

Dr. Zilva, of the Lister Institute, London, arrived at similar conclusions. He studied the action of ultra-violet rays upon cod liver oil, and found that this oil was completely inactivated (that is, that it no longer contained any vitamine A in active or recognizable form) by being exposed to such rays for 6 to 8 hours. This inactivation was shown to be due to the formation of ozone (a modified and more active form of oxygen) by the action of the ultra-violet rays upon the oxygen of the air, and not to the direct action of the rays themselves. (References: F. G. Hopkins, Biochemical Journal, 14, 724, 1920; J. C. Drummond and K. H. Coward, Biochemical Journal, 14, 734, 1920; S. S. Zilva, Biochemical Journal, 14, 740, 1920.)