"Protein-free milk" was prepared by acidifying with acetic acid, milk which had been freed from fat by centrifuging. The resulting whey, after the removal of the casein which flocks out on the addition of acid, was next heated to boiling to coagulate the lactalbumin. These are the principal proteins of milk, and with their removal about 93 per cent of the total nitrogen contained in the milk is abstracted. The solution which was filtered free from lactalbumin was evaporated to dryness, leaving a yellowish residue consisting of milk sugar, most of the salts of the milk, and a certain amount of nitrogenous compounds for the separation of which no method is known.

Osborne and Mendel held the view, which their own studies finally showed to be untenable (32), that the amount of protein or its equivalent, resulting from the digestion of milk protein or directly secreted by the mammary gland, which was contained in their "protein-free milk" was too small to be a factor of significance in their experiments. The final product they included in their diets contained about 0.7 per cent of nitrogen (30), which would be equivalent, if it were all in the form of protein or of its digestion products, to 4.37 per cent of dry matter. It is well known that some urea, purins, creatin, and other simple nitrogenous substances which circulate in the blood are always found in milk. It is certain, however, that there is a considerable amount of nitrogen in "protein-free milk" in forms yielding amino-acids in the body. Its property of enhancing certain incomplete proteins makes this view imperative.

From what has been said in Chapter II (A Biological Method For The Analysis Of A Foodstuff. 17. Mccollum'S Experiments Not Verified By Osborne And Mendel) it will be clear that a protein which is of low biological value for growth because of the fact that one or more of the digestion products which it contains is present in too small an amount to make possible its efficient transformation into body proteins, may be greatly enhanced in growth promoting value by the addition of the missing amino-acid or acids. This has been illustrated in the case of gelatin and for the meat digests prepared by Abderhalden (p. 61). It will be readily understood, therefore, that when a series of purified proteins was fed to young animals, the remainder of the diet consisting of 28 per cent of "protein-free milk," together with non-nitrogenous nutrients similar to the following:

Protein ................... 18.0 per cent.

"Protein-free milk" ............. 28.2 " "

Starch .................... 20.8 " "

Agar-agar.................. 5.0 " "

Fat ..................... 28.0 " " the amino-acids furnished by the "protein-free milk" may improve the quality of some or all of the pure proteins, and so change the experimental results as to make it unwarrantable to draw any conclusions concerning their relative nutritive values when fed without such an amino-acid supplement.