REPORT ON THE SEPARATION OF MEAT PROTEIDS. By F. C. Cook, Associate Referee. Samples of commercial meat extract and the following letter of instructions were sent to the seven collaborators, who expressed a willingness to cooperate on meat proteids this year: SEPTEMBER 4, 1907. DEAR SIR: I am sending you, under separate cover, a sample of meat extract. The sample should be kept cool until ready for work, then opened and well mixed. DIRECTIONS. Total nitrogen. (1) Determine the total nitrogen in not over 0.5 gram sample. Soluble nitrogen. (2) Dissolve 10 grams of the extract in water and make up to 500 cc. Filter on a plain filter paper, pouring back the first 10 or 20 cc. This is filtrate A. Determine the soluble nitrogen in 25 cc of filtrate A. For the acidity determinations use two 10 ce portions of filtrate A. In one case use phenolphthalein as indicator, diluting the solution before titrating. In the second case employ delicate litmus paper, testing by taking a drop outside by means of a small capillary tube. In determining the coagulable proteid take two 50 cc portions of filtrate A. To No. 1 add 9 grams of sodium chlorid, heat to boiling, add 1 cc N/10 acetic acid and boil three minutes. Let stand on steam bath ten minutes, filter, and wash with 50 cc hot water. Heat No. 2 to boiling and add 1 cc of N/10 acetic acid, boil three minutes, let stand on steam bath ten minutes, filter, and wash with 50 cc hot water. In both cases determine the nitrogen on the filter paper. Use nitrogen-free filter paper. The amido nitrogen is determined by placing 25 ce of filtrate A in a 100 cc flask. Add 15 grams of sodium chlorid and 25 cc of water. Shake and keep cool (15° C. or less) for two or more hours. Make a 24 per cent tannic-acid solution, filter and keep at same temperature. Add 30 cc of the 24 per cent tannic-acid solution to the flask, fill to mark with water, shake, and keep cool (15° C. or less) for twelve hours (usually overnight). Filter off 50 cc of the solution and determine nitrogen therein by adding a few drops of sulphuric acid and evaporating to dryness on the steam bath with help of vacuum if at hand. Add 25 or 30 cc of sulphuric acid but no potassium sulphate and proceed as in the Gunning method. It is necessary to run a blank, as the tannic acid often contains nitrogen. The nitrogen figure minus the blank nitrogen figure multiplied by 2 equals amido nitrogen in the filtrate from 25 cc of the original sample (0.5 gram). Kreatinin determination. Coagulate 20 cc of filtrate A, as in No. 2 under coagulable proteid, filter, and wash with hot water. Place the filtrate in a 500 ee flask, add 15 cc of saturated picric acid solution and 5 cc of 10 per cent sodium hydroxid, shake well. After standing 5 minutes dilute to mark with water and compare the color with an N/2 potassium dichromate solution in a Dubose colorimeter, the scale being set at 8 mm; 81 divided by reading x on the other scale equals milligrams of kreatinin. This is a modification of Folin's method as applied to the estimation of kreatin and kreatinin in the urine. For the determination of kreatin use 15 cc of filtrate A, coagulate as above, filter, and wash. Place in an Erlenmeyer flask. Add 5 ce of N/2 hydrochloric acid and attach a reflux condenser, place on steam bath for 34 hours, cool, transfer to a 500 cc flask, add 5 ce of N/2 sodium hydroxid and proceed as above, adding 15 cc picric acid, etc. This reading gives the total kreatinin. From the total kreatinin subtract the original kreatinin and multiply the difference by 1.16 to obtain the kreatin originally present. Report results as follows: Total nitrogen, per cent. Soluble nitrogen, per cent. Amido nitrogen, per cent. Kreatin, per cent. a Zts. physiol. Chem., 1904, 41: 223; Amer. J. Physiol., 1905, 13: 48. Acidity (1) using litmus cc N/10 sodium hydroxid per 100 grams. Coagulable nitrogen, using sodium chlorid, per cent. Duplicate determinations should be made. Any criticisms and suggestions will be gladly received. DISCUSSION OF RESULTS. In the sample sent out there was but a small amount of insoluble proteid present, consequently no conclusion can be drawn under that head. The cooperative results as given in Table I show a wide variation for total nitrogen, and consequently for the different nitrogenous constituents, as the nitrogen is the basis of the determination. The tannin-salt method gave fairly satisfactory results, although there is still considerable variation in the results obtained by the different collaborators. The trouble often experienced by the foaming in the Kjeldahl process, due to the presence of the tannin, is eliminated by evaporating the solution, after adding 2 or 3 drops of sulphuric acid, almost to dryness, then adding about 30 cc of sulphuric acid and no potassium sulphate, and digesting as usual. The proteose and peptone figures reported were obtained by difference. Cooperation was also obtained on the determination of coagulable proteids. There has been considerable discussion as to the use of sodium chlorid for this purpose, and the collaborators were instructed in one case to add 9 grams of sodium chlorid to 50 cc of the meat extract solution, while in the other case no sodium chlorid was to be added. As would be expected, practically all of the results were higher when sodium chlorid was used, but as the amount of coagulable proteid present in the sample was small the results are not striking. In Table II some results obtained by the referee are given, showing the influence of varying amounts of sodium chlorid on the coagulation of proteid in a commercial meat juice. TABLE II.-Coagulable proteid tests using varying amounts of sodium chlorid and 50 cc of the meat extract solution. These figures show that the amount of nitrogen precipitated increases with the amount of sodium chlorid used, and Mr. Lowenstein, chemist for Morris & Co., Chicago, has obtained similar results for coagulable proteids on samples of meat extracts. It is evident, therefore, that to obtain correct figures for coagulable proteid nitrogen no sodium chlorid should be employed. The two indicators, phenolphthalein and litmus, used in determining the acidity of meat products were compared, and the results obtained by the various chemists show that in practically all cases higher results were obtained where phenolphthalein was used. In a good many cases the difference is marked. The reason for this is that many of the organic acids have no action on litmus, but do act on phenolphthalein. The referee finds that phenolphthalein inevitably gives higher results than litmus, is easier to handle, and the end point is more accurate, but in certain dark-colored solutions of meat preparations litmus paper must be used. In Table III the results of acidity tests on solutions of meat extract and on similar solutions after removing the insoluble proteid by filtering, and after coagulating and filtering, are given: TABLE III.-Acidity determinations. [Comparing the original solution, the solution after filtering, and the solution after coagulation and filtering, using two indicators.] As the amount of insoluble proteid present in most of the samples was small, the results on this point are not conclusive, but the two latter sets of figures show lower results than those obtained by titrating the original solution, and in two cases the removal of the coagulable proteid before titration gives the lowest results. This is true only when litmus is used, the phenolphthalein results showing but little variation among themselves. As the amount of proteid present in meat products affects the acid determination, a uniform procedure should be adopted. In the case of meat extracts, most of which contain little insoluble and coagulable proteid, the titration of the original solution is recommended. The determination of kreatin and kreatinin was also investigated this year. According to the method suggested for kreatinin 20 cc of the coagulable proteid filtrate are used for the determination, but as this filtrate has previously been treated with 1 cc of N/10 acetic acid and boiled three minutes, the heat and acidity employed may have changed some kreatin to kreatinin, thereby giving too high results, consequently it is better to apply the kreatinin test to the original solution. This is satisfactory in the case of most meat extracts, as they contain little insoluble proteid. The effect of the presence of a considerable amount of proteid on the kreatinin test has not been determined. The best method of applying to meat products, especially meat extracts, the kreatin test, including an estimation of both kreatin and kreatinin in the form of kreatinin, has not been evolved. There are several points which remain to be settled. Folin originally applied his method to 10 cc of urine. In the method on which cooperation was requested a much larger volume of the solution was used. Undoubtedly the volume has some influence on the results, and this point needs investigation. Hehner in a recent article claims that the addition of 15 cc of picric acid is not sufficient, and that, upon adding 25 or 30 cc, higher and more accurate results are obtained. This point was investigated, using a solution of pure kreatin and a meat extract, and the addition of 30 cc of picric acid gave but slightly higher results than when 15 cc were used. The question of the amount of acid to be added for the conversion of kreatin into kreatinin, as well as the period of heating, are important factors. The referee has experimented with the cooperative method which is given in the letter of instruction, with the method as outlined by Grindley and Wood, and that used by Armour & Co., which is as follows: Make the volume of the solution up to 25 cc, place in a 6-inch porcelain evaporating dish, add one-half its volume of normal hydrochloric acid, and evaporate nearly to dryness on the water bath. Add 25 cc of water and 12.5 cc of normal hydrochloric acid, and repeat the evaporation to dryness. Take up the residue in 25 cc of water, add 15 cc of picric acid and 5 cc of sodium hydroxid, let stand five minutes, and then rinse into a 500 cc volumetric flask. This method gives good duplicates and is short and simple. With Grindley's method the duplicates were not so good nor the results so high, and the cooperative method gives lower results than either of the two others. It is evident that three and one-half hours' heating on the steam bath is not sufficient to convert all the kreatin to kreatinin. A method using 10 ce of normal hydrochloric acid and heating four hours in a boiling-water bath gives results that are fully as high as those obtained by Armour & Co. While none of the methods for kreatin and kreatinin are as yet entirely satisfactory, the value of these two determinations in analyzing meats and meat products has been generally recognized. Benedict and Myers have employed an autoclave for the conversion of kreatin to kreatinin. By this method fifteen minutes suffice for the complete conversion. The referee has this method under investigation and recommends that the study of an official method for kreatin and kreatinin be continued next year. с a Pharm. J., 1907, 78: 683. J. Biol. Chem., 1907, 3: 49. Amer. J. Physiol., 1907, 18: 397. One of the important features of this work is the criticism which it calls forth. The comments of the collaborators, whom the referee here takes occasion to thank for their helpful interest in the work, are as follows: COMMENTS BY ANALYSTS. H. S. Grindley, of Urbana, Ill., says: First, I do not think that the operation of determining the soluble nitrogen is of sufficient value to justify the additional labor. Second, I think there is serious objection to the determination of kreatinin in the filtrate obtained after the coagulation of the solutions by heat and acetic acid. It is my opinion that by such an operation kreatin will be converted into kreatinin. Investigations made in this laboratory conclusively prove that the kreatin existing in water extracts of flesh are very readily, in part at least, converted into kreatinin even in the presence of the neutral acidity of the extract of flesh. Third, the filtration of the solutions is very slow and tedious, and we have found that unless three or four filters are used for each solution there is an error in all determinations upon the filtrate due to the evaporation of the solutions during filtration. Fourth, since the filtration of a water solution of meat extract is so difficult, it appears that, as far as possible, determinations should be made directly upon the original solution without filtration. Our work demonstrates that the presence of proteids, even in considerable quantity, does not interfere with the kreatin and kreatinin determinations. Fifth, the acidity of ordinary extracts can be determined as well upon the solution of the extract before as after filtration. However, we have not found any meat extract which contains any considerable quantity of insoluble matter. Sixth, the titration of the solutions of the extracts when using litmus as an indicator was very unsatisfactory. Men in this laboratory working independently have obtained such results when using litmus as to indicate clearly the unreliability of this indicator. For these reasons, no results of acidity of meat extracts when litmus is used as the indicator are reported. Seventh, the tannin-salt method worked very nicely as to details of manipulation, and the duplicate results have been very satisfactory. On the other hand, it seems very unfortunate that a reagent which involves such a large correction factor must be employed. Although we have used Kahlbaum's very best tannin, it contained a large quantity of nitrogen. Arthur Lowenstein, of Chicago, Ill., makes the following suggestions and criticisms regarding the method : Total and soluble nitrogen: The methods for these determinations are quite satisfactory providing the sample is absolutely uniform, a difficult matter to accomplish with solid extracts containing 24 per cent moisture or less, and when only 0.5 gram of extract is employed an error is apt to be introduced. Acidity: With the majority of extracts it is preferable to employ phenolphthalein as indicator. The reaction with litmus is not very sharp and with very highly colored extracts, such as are frequently found, the use of an outside indicator is necessary. Coagulable nitrogen: The extract submitted contained practically no coagulable matter. However, in other samples the addition of sodium chlorid gave a higher result in every case. Amido nitrogen: The method of evaporation is slow, and equally good results were obtained if 50 cc of the filtrate were used after precipitation with tannic |