Liebig was also the father of the modern methods of organic analysis, and with him began the great accumulation of knowledge concerning the chemistry of the carbon compounds, including many products of the animal economy. These discoveries gave the world a knowledge of the constitution of foods, of urine, of feces, and of tissues, which was not possessed by Lavoisier.

Liebig applied to the problems of biology the mental wealth of the newer chemistry which he himself was creating. He knew that protein contained nitrogen, and in 1842 he suggested that the nitrogen in the urine might be made a measure of the protein destruction in the body.1 Bidder and Schmidt2 were the first to make systematic experiments upon this subject. They gave meat to dogs and cats and found that almost all the nitrogen contained in the meat was eliminated in the urine and in the feces. They3 make the following striking statement, which rings quite true to modern thought concerning protein metabolism: "Almost all the nitrogen of protein and collagen is split from its combination and carries with it enough carbon, hydrogen, and oxygen to form urea; the remaining part, containing five-sixths of the total heat value of the protein, undergoes oxidation to carbon dioxid and water which are eliminated in the respiration, the calori-facient function having been fulfilled." The results obtained by Bidder and Schmidt were attacked and were not finally established until proof was afforded by Carl v. Voit,1 who established the fact that an animal could be brought into what he called nitrogenous equilibrium. In this condition the nitrogen of the protein eaten was equal to the nitrogen eliminated from the body in the urine and feces. Thus Voit2 fed a dog for fifty-eight days with 29 kilograms of meat containing 986 grams of nitrogen, and found 982.8 grams of nitrogen in the excreta of the period. The amount of N in the urine was 943.7 grams, and in the feces 39.1 grams. The difference between the amount of nitrogen ingested and that recovered in the excreta was only 3/10 of 1 per cent. It therefore seemed extremely probable that the excretory outlet for protein nitrogen was in the urine and in the feces and that other sources of its loss were normally negligible. But in order to establish the fact it was necessary to consider the following questions:

1 Liebig: "Die organische Chemie in ihrer Anwendung auf Physiologie und Pathologic," 1842.

2 Bidder and Schmidt: "Die Verdauungssafte und der Stoffwechsel," 1852, pp. 333, 339.

3 Bidder and Schmidt: Ibid., p. 387.

Is the nitrogen of the air built up into organic compounds within the body? Is any protein nitrogen given off as nitrogen gas? As ammonia gas? In the sweat? How much is lost through the growth of the hair, nails, and epidermis?

Lavoisier" had said that nitrogen gas had nothing to do with respiration. Regnault and Reiset3 sometimes found that animals under a bell-jar absorbed nitrogen gas and at other times gave it off. The quantity in both cases was extremely small and can be explained by slight errors in gas analysis due to inexact temperature records. Regnault and Reiset found no measurable quantities of ammonia or of sulphur-containing gases in the expired air, and they discovered that hydrogen might replace nitrogen in the atmosphere without affecting the course of metabolism.

1 Voit: "Physiol. Chem. Untersuchungen," 1857.

2 Voit: "Zeitschrift fur Biologie," 1866, ii, 35.

3 Regnault and Reiset: "An. de chimie et phys.," Paris, 1849, Sec. 3, Tome xxvi.

The experiments of Bachl1 showed that a rabbit with a tracheal cannula could be made to expire for six hours through Nessler's reagent without the indication of a trace of ammonia in the breath. This has also been shown after making an Eck fistula in a dog,2 where there is an increase in the amount of ammonia in the blood and in the urine. The lungs are not permeable to ammonia.3 The ordinary insensible perspiration is not accompanied by any appreciable loss of nitrogenous excreta, although profuse sweating certainly brings out some urea, uric acid, and other nitrogen extractives normally excreted in the urine. The experiments of Benedict4 show that the cutaneous excretions of a resting man may amount to 0.071 gram nitrogen per day; of a man at moderate work to 0.13 gram per hour, and at hard work for four hours to 0.22 gram per hour.

Voit6 collected the hair and epidermis from a dog for 565 days and found an average daily output of 1.2 grams with 0.18 gram of nitrogen. Moleschott6 cut the hair and nails of several men once a month. The daily outgrowth of hair was 0.20 gram with 0.029 gram of nitrogen, and of nail substance 0.005 gram with 0.0007 gram of nitrogen. The waste through the human epidermis has not been measured, but it must be very slight. The above sources of error were thus shown to be negligible.

The view that the nitrogen of the urine and feces could be made a measure for the determination of protein metabolism was thus securely established. Urea, the principal nitrogenous end-product derived from protein, was therefore shown to be not an adventitious product, but one normally proportional to the protein destruction. It was known that meat protein in general contained about 16 per cent, of nitrogen, or 1 gram of nitrogen in 6.25 grams of protein. Therefore for every gram of nitrogen found in the excreta, 6.25 grams of protein have been destroyed in the body. It is evident that if protein nitrogen be retained in the body a new construction of body tissue is indicated, whereas if more nitrogen is eliminated than is ingested with the food, a waste of body tissue must take place. The discovery of the method of calculating the protein metabolism led Voit to suggest to Pettenkofer that he construct an apparatus with which the total carbon excretion might be measured, including that of the respiration as well as that of the urine and the feces. Voit saw that with these data it would be possible to determine just how much of each food-stuff was actually burned in the human body. He has described the delight which he and Pettenkofer experienced when their wonderful machine began to tell its tale of the life processes. The cost of the apparatus, which was considerable, was defrayed by King Maximilian II of Bavaria.

1 Bachl: "Zeitschrift fur Biologie," 1869, v, 61.

2 Salaskin: "Zeitschrift fur physiologische Chemie," 1898, xxv, 463.

3 Magnus: "Archiv fur ex. Pathologie und Pharmakologie," 1902, xlviii, 100.

4 Benedict: "Journal of Biological Chemistry," 1906, i, 263.

5 Voit: "Zeitschrift fur Biologie," 1866, ii, 207. 6 Moleschott: "Untersuchungen zur Naturlehre," xii, 187.