It has been frequently demonstrated that it is impossible to secure growth or maintenance if the inorganic constituents of the diet are improperly adjusted.51a The most satisfactory mixture of salts to supplement a salt-free diet attained to by Osborne and Mendel in their early experiments, after many attempts to modify the relative proportions of the differentiations present, was essentially that used by Rohmann.52

Salt Mixture I (Rohmann And Osborne And Mendel)

2.5 per cent of this was incorporated in their salt-free rations.

Having observed the excellent results attained with rats fed on a food mixture of milk powder (60%), starch (16.7%), and lard (23.3%), in which the inorganic salts were obtained entirely from milk, the idea suggested itself that milk freed from fat and protein might be used with advantage as the source of inorganic salts. Accordingly they prepared a product designated "protein-free milk," as follows: 53

51a Scala (Ann. igiene 99, 215, 286,1010; Expt. Sta. Record 43, 402) advances the hypothesis that deficiency diseases originate in a deficient mineral nutrition, either in the lack of certain acids or bases or of complexes by means of which they are transported. In scurvy the inorganic substance which is lacking is thought to be phosphates of the earth metals, which exist in food materials in combination with organic material in the form of complexes, easily decomposed by heat and by desiccation. The destruction of these complexes tends to bring about a state of acidosis and prevents the transportation of calcium phosphate, etc., to the bones. In beriberi the alkali phosphates are unable to reach the central nervous system through the destruction of the organio complexes containing them and the development of a form of acidosis. The fact that various mineral substances when added to a beriberi-producing diet fail to bring about appreciable benefit is explained on the ground of inability to reproduce the exact complexes found in the original food material.

Loew (Vierteljahrs. ger. .Med. 61, 151, 1021) contends that calcium equilibrium depends on the nature of the diet. A high-fat diet means a low calcium assimilation. A decrease in the alkalinity of the blood means a loss of calcium by excretion. A prolonged under-supply of calcium gives rise to diverse pathological conditions. (See also, Rubner, Vierteljahrs. ger. Med. 61, 155 1021).

52 Jahresb. f. Thier. Chem. 38, 650, 1000.

Perfectly fresh centrifugated milk, nearly free from fat, was precipitated in lots of about 36 liters by diluting with 7 liters of distilled water which contained 164 cc. of concentrated hydrochloric acid. The flocculent precipitate of casein was strained out on cheesecloth, and the very nearly clear solution was filtered through a pulp filter. The filtrate, which at the most was very slightly turbid from suspended fat, was tested carefully by the alternate addition of dilute alkali and acid to determine whether any more casein could be separated from it. The addition of alkali caused a slight precipitate which did not increase on adding more alkali or dissolve on the addition of even relatively large amounts of alkali. This was presumably chiefly calcium phosphate. The addition of acid in no case caused any further precipitation. The filtered milk serum was then heated to boiling for a few minutes and filtered. The filtrate, which was in all cases water clear, was then neutralized to litmus with a dilute solution of sodium hydroxide and evaporated to dryness on a steam bath at a temperature of about 70o C. The product thus obtained formed a friable, pale yellow mass which was easily reduced to a fine powder by grinding in a mill.

With this preparation, as has been already pointed out, they obtained results successful beyond their expectations; results which have been confirmed by later investigations.54

Considerable discussion arose as to the amount and effect of the nitrogen remaining in this "protein-free milk." In their original communication these investigators say:

Several grama of this powder were tested for protein by dissolving in about 30 cc. of water containing a little hydrochloric acid and warming gently. The solution was then saturated with ammonium sulphate. The precipitate, which appeared to consist chiefly of calcium sulphate was separated by centrifugation, dissolved in a little water, and potassium hydrate solution and copper sulphate added. The solution showed no evidence of the biuret reaction until it was saturated with potassium hydroxide and shaken with alcohol. It then separated into two layers, the upper alcoholic layer showing a slight but positive biuret reaction. Millon's reaction tried on portions of two or three grams of the substance did not give a positive reaction. Nitrogen determinations in several lots of the protein-free milk powder thus made showed them to contain 0.66, 0.59, 0.60, 0.72, 0.71, 0.67, 0.75, per cent of nitrogen. Munk 55 states that if the proteins of milk are precipitated by alcohol, or separated according to Hoppe-Seyler, from one-thirtieth to one-fifteenth of the protein remains dissolved. All the proteins can be precipitated only by tannin in the cold or by copper hydroxide on heating. He further states that cow's milk contains about one-sixteenth of its nitrogen in non-protein form.

Since our protein-free milk powder was equal to 60 per cent of the total solids of the milk, it should, if Munk's statements are correct, contain 0.48 per cent of non-protein nitrogen, thus leaving at the most only 0.28 per cent of the protein nitrogen, equal to 1.69 per cent of protein. Since 100 grams of the food mixture employed in our experiments contained 282 grams of protein-free milk powder, we can assume that at the most the food pastes thus made contained only 0.48 per cent of milk protein. The protein-free milk powder thus produced as above described left about 14.5 per cent of inorganic matter on ignition. This includes not only the inorganic constituents of the milk, although by no means in the combination in which they occur in the mammary secretion, but also the inorganic salts when were formed by the addition of the hydrochloric acid used to precipitate the casein and also the sodium salts which resulted from neutralising the milk serum with sodium hydroxide solution.

53 Osborne and Mendel, Cam. Pub. 166, Pt. 2, p. 80-81. 54 Wheeler, J. Exper. Zool. 15, 209, 1913; Mitchell and Nelson, J. Biol. Chem. 23, 461.

55 Munk, Virchow's Archiv. f. path. Anat. 134, 601, 1893.