Osborne, Mendel and McCollum went even further. They showed that while the fat from egg yolk and codliver oil, and, to a certain extent, beef fat, could successfully replace butter fat, neither olive nor almond oil could; so that the latter two were no better than lard. And what was very significant in these experiments was that by the substitution of the vitamine-containing fat for the fat which evidently was vitamine-free, not only did the rats resume growth and gain in weight, but disorders due to malnutrition disappeared, and the general resistance of the rats to disease increased.

That the deficiency of lard is not due to its manner of preparation, which involves steaming, and which might perhaps destroy the vitamine, was shown by Drs. Osborne and Mendel by heating butter fat with steam - an operation which did not make the butter fat any the less effective. Incidentally this indicated a surprising resistance of vitamine to the effect of high temperature. Experiments to be described presently will necessitate a modification of this view - at least, in so far as vitamines other than the one just described are concerned.

Vital to the organism as these vitamines appeared to be, they could not serve the place of a deficient supply of fat, protein, carbohydrate and mineral salts. It is of the utmost importance to remember this statement.

For some time the results of our American investigators were taken at their face value, and the opinion was generally expressed that the vitamine, though not actually in the hands of the scientific police, was known to be hiding in the fat portion of the food.

Conflicting Observations

Certain clinical observations on such diseases as beriberi, to be discussed in the next chapter, also strongly supported the view that vitamine is necessary; but there was one sharp point of difference between the two schools. McCollum, Osborne and Mendel had repeatedly made the observation that butter fat is extremely rich in vitamine; but Funk now showed that butter fat had no power to cure beriberi, which, as will be shown later, is a disease due to vitamine deficiency. What was to be made of such conflicting observations? Were the experimenters at fault, or was there more than one vitamine?

Dr. McCollum Solves the Difficulty. The problem was attacked by Dr. McCollum. A synthetic diet which he had found amply sufficient to meet all requirements consisted of the following: casein (the protein obtained from milk) 18 per cent; lactose (the scientific name for milk sugar, which belongs to the class of carbohydrates and is obtained from milk) 20 per cent; butter fat 5 per cent; salt mixture (meaning the mineral salts) in quantity and variety such as is found in milk, and proved to be superior to Dr. Hopkins' original salt mixture; and starch (another carbohydrate) to make up 100 per cent. Water of course was offered a-plenty: as our scientist friends, fond of their Latin, are in the habit of putting it, ad libitum.

Polished rice, a term to be explained in the next chapter, was known to produce beriberi in men and similar diseases in birds. Rice, like all grain products, contains a large percentage of carbohydrates, chiefly starch; the polished variety as high as 80 per cent. McCollum now modified his synthetic diet in such a way as to add rice to it, but subtract the starch and milk sugar; so that the proposed diet consisted of rice, casein, butter fat and salt mixture. He reasoned that the rice contained more than enough starch to take the place both of the starch and the milk sugar in the original diet. But with this modified diet his animals developed beriberi symptoms.

This was rather perplexing. The experiment confirmed Funk's assertion that butter fat could not cure the disease, for the diet included ample proportions of this fat. On the other hand, butter fat in the original synthetic diet, had been shown to be the cause of renewed growth and general development in rats.

McCollum very carefully went over his data. The only substance that was not included in his modified diet that had formed part of his original synthetic diet was milk sugar. He had no reason to believe that milk sugar as a carbohydrate was in any way superior to starch. But perhaps after all it was? Or perhaps, still more likely, this sugar contained vitamine, not necessarily identical with the vitamine in butter fat, which had the power of curing beriberi, just as the butter fat vitamine had the power of promoting growth?

And truly enough, when milk sugar was included in the diet of the sick animals they very soon recovered.

McCollum next showed that by very careful purification of the milk sugar he obtained a product which could no longer cure beriberi. In other words, the purer the milk sugar the less efficacious the remedy! This clearly pointed to but one thing: that the cure was not due to the milk sugar, but to some impurity in milk sugar; and that this impurity could be removed by carefully refining the sugar.

That such a view was sound was proved when to a diet including the purified milk sugar, which had no curative power, the washings containing all the impurities that had been removed, were added; the cure was immediate and effective.

How small in quantity the vitamine present as an impurity in the lactose actually is, may be judged by the following: The milk sugar used by Dr. McCollum was manufactured by Kahlbaum, a concern renowned in the scientific world for the purity of its chemicals. After further treatment by McCollum, chemical analysis failed to show any material difference between the original and the treated sugar. Yet something must undoubtedly have been removed, for, biologically, the two sugars acted so differently; and to escape detection, this something - our vitamine - must have been present in exceedingly minute proportions.

Two Distinct Vitamines

Clinicians and physiologists had arrived at one conclusion: a factor other than protein, fat, carbohydrate and mineral salts is necessary. This factor they called vitamine. But McCollum now showed that there are in fact two distinct factors, two distinct vitamines at least, and that "the substance which relieves the condition of polyneurites in pigeons (comparable to beriberi in man) is always present in preparations which render the purified food mixture capable of promoting growth."

This last statement is very important. Says McCollum, if you think that in our earlier experiments with butter fat versus lard the only thing that mattered is the vitamine in the butter fat, you are much mistaken; there was the other vitamine, present in my milk sugar (and as we shall show, in other food products) that played an equally important part; for it prevented beriberi. It was there, but you and I were not aware of it.