B Y THE use of paper chromatography, Block, Horwitt, and Bolling' showed qualitatively that human enamel eukeratin contained most of the known amino acids, and gave quantitative values for arginine, histidine, and lysine.Losee and Hess,2 who had prepared the protein analyzed by Block, Horwitt, and Bolling, determined cystine, methionine, and phenylalanine by colorimetric methods and subsequently3 also determined arginine, histidine, and lysine, and confirmed the statement that the normal enamel protein is an eukeratin. However, the sum of the values for these six amino acids accounts for less than 15 per cent of the total nitrogen of the protein. In order to characterize completely the protein and also to permit comparison with dentinal protein and other eukeratins, we have estimated the other amino acids in normal enamel protein. EXPERIMENTALThe enamel protein was prepared as described by Losee and Hess.2 The nitrogen content was 13.59 per cent and the ash content was 9.59 per cent, based upon the weight of the desiccator-dried sample. The amino acids, except the six previously determined colorimetrically, were all estimated by the microbiologic methods used by Horn, Jones, and Blum.4 The inoculumns were prepared using the media of Hac, Snell, and Williams5 because the liver infusion used by Horn, Jones, and Blum4 was unavailable. Leuconostoc mesenteroides was employed for the assay of leucine and isoleucine, and Streptococcus faecalis for threonine and valine as recommended by Horn, Jones, and Blum.4 Leucine, isoleucine, valine, glutamic acid, aspartic acid, proline, glycine, and tyrosine were determined, as suggested by Neuman,6 using Leuconostoc mesenteroides and serine employing Streptococcus faecalis. For each amino acid from three to four concentration ranges and triplicate determinations for each concentration were made, of the resulting lactic acid using 0.05 N NaOH and bromthymol blue as the indicator. The results were averaged.The protein was hydrolyzed with 20 per cent hydrochloric acid for six hours at 125°C., and the hydrolysate was freed from excess acid by evaporation under reduced pressure. The results of these analyses and the values for the six amino acids previously determined are given in Table I, together with the range of values for the amino acids in other eukeratins as cited by Block and Bolling.7 The nitrogen content of the amino acids determined accounts for 98.3 per cent of the total nitrogen of the protein.

THE rather meager literature on the lipides in teeth has been reviewed previously by Leopold, Hess, and Carter.' These investigators also reported that human dentin contains 0.024 per cent cholesterol. The present paper deals with the determination of cholesterol in enamel and the total lipide content of both enamel and dentin. EXPERIMENTAL Sample Preparation.-The dentin used was prepared in the same manner as that previously used for the isolation of cholesterol.1 The enamel was prepared by the method employed by Losee and Hess2 for the isolation of enamel protein.Cholesterol in Enamel.-The method of isolation of cholesterol from dentin that was most satisfactory was used for the isolation and estimation of the cholesterol in enamel. The procedure involved extraction of the enamel with an aqueous solution containing 30 per cent KCl and 1 per cent K2C03. Twenty milliliters of solution was used for each gram of enamel. After several extractions the combined extracts were treated with 0.4 Gm. of potassium acetate, 0.4 Gm. of silica gel, and 0.4 ml. of glacial acetic acid per gram of original sample. The extract was then shaken for 30 minutes, filtered with suction through a fine-sintered glass filter, and the filtrate was extracted with petroleum ether. Both the petroleum ether extract and the aqueous extract were analyzed for total cholesterol. To determine if the treatment with silica gel adsorbed any cholesterol, a recovery experiment using pure cholesterol was run in which 2 solutions, each containing 1.76 mg. of cholesterol in 100 ml. of H20, were used. One solution was treated with the same amount of silica gel used in the enamel extraction procedure and the other was used as a control. Cholesterol determinations gave a value of 1.76 mg. in the control and 1.43 mg. in the silica gel-treated solution, a loss of 18 per cent. Correction for this loss incurred in the procedure was used on the determinations of the cholesterol content of the enamel. All cholesterol determinations were made by the same method previously used.