ERRORS IN THE QUANTITATIVE DETERMINATION OF CHOLESTEROL BY RITTER S METHOD : THE INFLU- ENCE OF AUTOLYSIS UPON CHOLESTEROL.1

ERRORS IN THE QUANTITATIVE DETERMINATION OF CHOLESTEROL BY RITTER S METHOD : THE INFLU- ENCE OF AUTOLYSIS UPON CHOLESTEROL.1 BY HARRY J. CORPER. (From the Pathological Laboratory of the University of Chicago.)2 (Received for publication, December 3, 1911.) While carrying out analyses of normal and autolyzed spleens reported in previous papers3 the Ritter method4 for determining cholesterol quantitatively was found inadequate. The results were so variable for the amounts used that it was thought advisable to investigate the steps in the method in order to find the source of error, and alsd to find out whetheti the method actually could be used for the quantitative determination of cholesterol in tissues. At least one other investigator, Helen Baldwin,6 has had difficulty in the quantitative determination of cholesterol by this method. The quantitative methods for cholesterol determination suggested up to 1908 (a discussion of these methods is given by Glikir+) practically all depend either upon the saponification of the fat to be examined, the cholesterol being recovered as such, or upon the esterification of the cholesterol and the determination of the iodine figure or saponification number. More recently Windaus has suggested a new method for the determination of cholesterol and cholesterol esters, precipitating 1 This work has been aided by a grant from the Rockefeller Institute for Medical Research. 2 A portion of this work was done in the Physiological Laboratory of the University of Illinois. s Cf. preceding article on The Chemistry of the Dog s Spleen and reference 23, page 44. 4 E. Rittq: Zeitschr. f. physiol. Chem., xxxiv, p. 461, 1903. 6 Helen Baldwin: This Journal, iv, p. 213-219 (218), 1908. 6 W. Gilkin: Biochem. Centralbl., vii, pp. 289-306; 357-77, 1908. 7 A. Windaus: Zeitschr. f. physiol. Chem., Ixviii, pp. 110-117, 1910. 37

38 Cholesterol Determination them by means of digitonin. Lapworth* reports good results using this method. In order to more clearly understand where loss in Ritter s method might occur, the literature was searched for studies on the action of the various chemicals used in the method upon cholesterol in forming salts, or actual decomposition products and the properties of these compounds. Lindenmeyer prepared sodium cholesterolate, which he purified from chloroform by cooling the solution on ice, and found it to be insoluble in water, and that it was only slowly decomposed by water, weak alcohol hastening this decomposition. Obermtillerio prepared potassium cholesterolate and found the properties similar to those of the qod ium oompound. Darmst,%dter and Lifschutzll were able to obtain oxidation products of cholesterol by heating cholesterol with alcoholic potash with a reflux condenser for eight hours, the yield being 20 to 25 per cent. Lifschutzu divided the products into three phases: (1) OXYcholesterin ethers; ( 2) Oxycholesterins and (3) Dicarbonsiiure (Chollansaure), the first two being soluble in all the ordinary solvents except water, and the last being soluble in water and alkalies and precipitating as white flocks on acidification. Schulze and Winterstein noted a drop in the melting point of cholesterol exposed to the light,, apparently due to oxidation, as an atmosphere of CO, prevented this change. Lifschutz14 states that one hour s cooking with half-normal alcoholic potash does not alter cholesterol. E. Schulze15 recovered cholesterol from substances in which potash did not liberate it so that it could be extracted by ether, by heating with benzoic acid in sealed tubes and forming an ester insoluble in alcohol and ether. 8 A. Lapworth: Journ. of Path. and Back, xv, pp. 254-61, 1911. 9 Lindenmeyer: Erdmann s Praktische Chemie, xc, pp. 321-332, 1863. 10 Obermtiller: Zeithschr. f. physiol. Chem., xv, pp. 37-48, 1891. 11 L. Darmstlidter and J. Lifschutz: Ber. d. deutscl;. them. Ges., xxxi, pp. 1122-27, 1898. I2 J. Lifschutz: Zeitschr. f. physiol. Chem., 1, pp. 436-40, 1906-07. 13 E. Schultz and E. Winterstein: Ibid., xliii, pp. 316-19, 1995; xlviii, pp. 546-48, 1906, 14 J. Lifschutz: Ibid., lviii, p. 175, 1908. I5 E. Schulze: Zeitschr. f. anal. Chemie, xvii, p. 173.

Harry J. Corper 39 Very little reliable work has been done on the changes occurring in. cholesterol during autolysis, probably in part because of the fact that no reliable quantitative cholesterol method has been available, and partly because the change occurring, if such does occur, is naturally a slow one. Windaus,l j method for determining. cholesterol and cholesterol esters, and Lifschutz s17 recent investigations upon the deterrriination of the presence of oxidation products of cholesterol, should make research fruitful along these lines. Moore * failed to find any change in the cholesterol content of the liver in autolysis under toluene for forty-two days at 37 C. (Cholesterol analyzing 0.038 per cent before and 0.0372 per cent after autolysis). He also found no significant difference between the cholesterol content of a normal area (0.64 per cent) and an infarcted area (0.58 per cent) in a human spleen, and strongly objects to the reasoning of Carbone,ls who believes that cholesterol originates from lecithin by decomposition, and of WaldvogePO who claims to have established the same by digesting lecithin with sterile liver juice, and who also found an increased cholesterol content in pathological livers as compared to normal (normal cholesterol content being 0.42. per cent, pathological-acute poisoning-being 24.46 per cent according to these analyses.) EXPERIMENTAL PART. METHODS. Ritter puts 50 grams of fat into a p.orcelain dish, adds 100 cc. of alcohol, brings it to a boil on the water bath, and then adds 8 grams of sodium dissolved in 160 cc. of 99 per cent alcohol with constant stirring. (The sodium alcoholate is prepared according to the method described by Kossel and Kriiger.21 These authors bring the absolute alcohol to a boil under a reflux condenser and carefully add the metallic sodium to it while boiling. They state that 10 cc. of a 5 per cent sodium alcoholate 16 Windaus: lot. cit. 17 J. Lifschutz: Zeitschr. f. physiol. Chem., liii, pp. 140-48, 1907. I* F. Craven Moore: Medical Chronicle, xlvii, pp. 20440, 1907-08. IQ Tito Carbone: Arch. ital. de biol., xxvi, p. 279, 1896. 20 Waldvogel and Mette: Mfinch. med. Woch., liii, p. 402, 1906. Q1 A. Kossel and M. Kriiger: Zeitschr. f. physiol. Chem., xv, p. 321, 1891.

40 Cholesterol Determination. solution thus prepared will saponify 5 grams of mutton tallow, and 15 cc. will saponify 5 grams of butter fat).22 The alcohol is then evaporated off on the water bath and about one and one- -half times as much salt as fat used is added, and enough water so that most of the contents of the evaporating dish goes into solution. Th IS is then dried on the water bath with constant stirring, and then at 8O C, in a drying oven. It is pulverized, put into a sulphuric acid dessicator for a short time, then into an extraction thimble, and is extracted in a Soxhlet apparatus with ordinary ether for nine hours. The ether extract is then put into a separatory funnel and shaken out with water to remove glycerin. The ether extract is dried, dissolved in hot alcohol, precipitated by means of water, precipitate dried at loo-120 C. and weighed. Experiments on the Effect of Sodium Alcoholate upon the Quantitatitle Yield of Cholesterol by Ritter s Method. In order to test the loss occasioned by the steps in the method, the following experiments were carried out, using pure cholesterol instead of a complex fat mixture. EXPERIMENT 1. The amount of cholesterol which can be recovered from salt mixture, being mixed in alcohol solution, dried, and extracted by means of absolute ether in a Soxhlet apparatus, and the ether extract shaken out by means of water. (a) Used 0.1016 gram cholesterol and recovered 0.1026 gram. (b) Used 0.1020 gram cholesterol and recovered 0.1030 gram. EXPERIMENT 2. Amount of cholesterol which can be recovered from salt mixture after treating with sodium alcoholate, evaporating to dryness, dissolving residue in ether and shaken out by means of water. (a) Used 0.1057 gram cholesterol mixed with 5 cc. of 5 per cent sodium alcoholate and recovered 0.1048 gram cholesterol. (b) Used 0.1007 gram cholesterol mixed with 10 cc. of 5 per cent sodium alcoholate and recovered 0.1016 gram cholesterol. (c) Used 0.1005 gram cholesterol mixed with 40 cc. of sodium alcoholate and recovered 0.0950 gram cholesterol. (d) Duplicate of (c). Used 0.1003 gram cholesterol and recovered 0.1020 gram cholesterol. EXPERIMENT 3. Amount of cholesterol which can be recovered after solution in absolute alcohol, saponification by means of 10 cc. of sodium *2 The sodium alcoholate employed in the following experiments was prepared according to this method, 5 per cent strength being used.

Harry J. Corper 41 alcoholate, evaporated to dryness, mixing with salt (10 to 15 grams), and extraction by means of absolute ether in a Soxhlet for 9 hours, shaking out ether extract with water, etc. (a) Used 0.1013 gram cholesterol and recovered 0.0390 gram. (b) Used 0.1012 gram cholesterol and recovered 0.0437 gram. EXPERIMENT 4. Similar to Experiment 3 but using only 5 cc. of sodium alcoholate. (a) Used 0.1016 gram cholesterol and recovered 0.0847 gram. (b) Used 0.1042 gram cholesterol and recovered 0.0358 gram. EXPERIMENT 5. Similar to Experiment 3 but using 40 cc. of sodium alcoholate. (a) Poured on salt in absolute alcohol solution, after saponifying. Used 0.1014 gram cholesterol and recovered 0.0415 gram. (b) Duplicate of (a). Used 0.1016 gram cholesterol and recovered 0.0390 gram. (c) Evaporated to dryness after saponification and emulsionized by means of water, mixed with salt, dried, extracted with ordinary ether, etc. Used 0.1032 gram cholesterol and recovered none. (d) Duplicate of (c). Used 0.1046 gram cholesterol and recovered none. (e and f) Not evaporated to dryness after saponification, emulsionised with saturated salt solution, dried, ground up, extracted with absolute ether, etc. Used 0.1023 gram cholesterol and recovered 0.0163 gram. Used 0.1002 gram cholesterol and recovered 0.0150 gram. EXPERIMENT 6. Amount of cholesterol that can be recovered after saponification by means of 40 cc. of sodium alcoholate, evaporating as nearly dry as possible, dissolved in ether and in water and these extracts poured on salt and dried and then extracted in a Soxhlet by means of absolute ether, etc. (a) Used 0.1010 gram cholesterol and recovered 0.0770 gram.. (b) Used 0.1003 gram cholestero! and recovered 0.0700 gram. As a result of the above experiments we can conclude that even an excess of 5 cc. of 5 per cent sodium alcoholate added to cholesterol, will prevent its complete extraction from a dried salt mixture by means of ether. That the trouble lies in the use of an excess of sodium alcoholate is further shown by the following experiments: In order to simplify matters the following abbreviation is used for the various steps. A. Cholesterol dissolved in absolute alcohol and heated on water bath for three days. B. Saponifying with sodium alcoholate.

42 Cholesterol Determination C. Mixing with N&l and drying. D. Extracting in a Soxhlet with absolute ether. E. Shaking out the ether extract with water in a separatory funnel. F. Allowing the ether to evaporate off at room temperature, and drying the residue at 100 C and weighing. TABLE OF RESULTS. Cholesterol. A. F... E. F... A. C. D. E. F... B. C. D. E. F... A. B. C. D. E. F.... 0.1068 0.1075 0.1001 0.1013 0.1001 0.0969 0.1096 0.0955 O.iO61 / 0.0675 gram 0.00 0.00 0.0032 0.0141 0.0386 In the above experiments the exact amount of sodium alcoholate used was not noted, as they were carried out before the first set cited. Now if an excess of sodium alcoholate thus affected the yield of cholesterol by the Ritter method when pure cholesterol was used, what would be its effect, upon the cholesterol yield from tissues? In this case we are unable to tell the exact, amount of fats and esters present, and therefore the amount of sodium alcoholate necessary to saponify them. If we use too small an amount our result will be high, due to the unsaponified fats and esters remaining as such with the cholesterol; and if we use too much the cholesterol yield will be low. To test these points the following experiments were carried out in the ether and alcohol extract from a steer spleen. The steer spleen weighed about. 850 grams (moist) and yielded an alcohol. and ether extract weighing 29.13 grams, which was dissolved in a liter of absolute alcohol and divided in 50 cc. samples (5 per cent. of the total extract,) for the following analyses for cholesterol. Cholesterol. EXPERIMENT 1. The extract after evaporation was dissolved in 10 cc. of absolute alcohol and warmed, 10 cc. of 5 per cent sodium alcoholate was added and the mixture warmed several hours, evaporated to dryness, redissolved in absolute alcohol, poured on 10 to 15 grams of salt, dried, extracted in a Soxhlet with absolute ether.

Harry J. Corper 43 Results. (a) Yielded 0.0263 gram cholesterol. (b) Yielded 0.0195 gram cholesterol. EXPERIMENT 2. Identical with Experiment I except that the saponified mixture was poured directly on the salt (without evaporation). Results. (a) Yielded 0.0162 gram cholesterol. (b) Yielded 0.0380 gram cholesterol. EXPERIWENT 3. Identical with Experiment 2 except 30 cc. of absolute alcohol was used as a solvent before saponification. Results. (a) Yielded 0.0215 gram cholesterol. (b) Yielded 0.0528 gram cholesterol. EXPERIMENT 4. identical with Experiment 2, but used 40 cc. of sodium alcoholate and evaporated the saponified mixture as nearly to dryness as possible, redissolved and poured on salt. Results. (a) Yielded 0.0072 gram cholesterol. (b) Yielded 0.0066 gram cholesterol. (c) and (d) were not evaporated after saponification before adding to the salt. (c) Yielded 0.0050 gram cholesterol. (d) Yielded 0.0100 gram cholesterol. EXPERIMENT 5. identical with Experiment 1 except that only 5 cc. of sodium alcoholate was used. Results. (a) Yielded 0.1773 gram cholesterol. (b) Yielded 0.1600 gram cholesterol. EXPERIMENT 6. The object of this experiment was to compare an old preparation of sodium alcoholate (five months old) and of dark brown co101 with the freshly prepared compound. It is practically a duplicate of Experiment 5 but using 5 cc. of old sodium alcoholate. Results. (a) Yielded 0.1624 gram cholesterol. (b) Yielded 0.1840 gram cholesterol. EXPERIMENT 7. Resembled Experiment 5 except in that the mixture was not evaporated to dryness after saponification but poured directly on the salt after standing 24 hours. Results. (a) Yielded 0.1306 gram cholesterol. (b) Yielded 0.1312 gram cholesterol. EXPERIMENT 8. identical with Experiment 1 but used 3 cc. 5 per cent sodium alcoholate. (The resulting product was only slightly oily in appearance). Results. (a) Yielded 0.2180 gram cholesterol. (b) Yielded 0.2155 gram cholesterol. EXPERIMENT 9. Identical with Experiment 8, but used only 1 cc. of sodium alcoholate. (The product was not crystalline but oily). ResuZts. (a) Yielded 0.3543 gram cholesterol. (b) Yielded 0.2956 gram cholesterol. As a result of the above experiments we can conclude that the best yield of cholesterol is obtained from the alcohol and ether

Cholesterol Determination extract of the spleen when about 5 cc. of 5 per cent sodium alcoholate is used to saponify 1.5 gram of the ether-aclohol extract; we mu&, however, expect an error in the quantitative results on this amount of extract of from 10 per cent to 25 per cent. Cholesterol in Autolysis. Remembering the possibility for analytical error by theritter method as shown above, the following experiments can carry no great weight, but will merely be cited to show that there is no marked change in the cholesterol content of the spleen (dog) during autolysis. EXPERIMENT 1. Sixty-five grams of ground dog spleen tiere mixed with 0.338 gram of cholesterol suspension (made by dissolving the cholesterol in a minimum amount of absolute alcohol and pouring it into 0.9 per cent sodium chloride solution), toluene was used as preservative and the mixture allowed to autolyze for ninety-two hours at room temperature. Recovered 0.591 gram of cholesterol or 0.253 gram above the amount added which must have come from the spleen. (Extraction, etc., was carried on here as in the case of the steer spleen analyses.) EXPERIMENT 2. Sixty-five grams of ground up dog spleen was mixed with 0.314 gram cholesterol (suspended in 0.9 per cent NaCl-toluene water). The mixture autolyzed at room temperature for fifty-three hours. Recovered 0.5566 gram cholesterol or 0.242 gram of cholesterol over the amount added. As a few successful analyses for cholesterol were obtained while carrying out the autolysis experiments reported in previous papers= they may be put into tabulated form for comparison with the two above mentioned experiments. For the sake of convenience the figures will be given in the form of the amount of cholesterol found in one gram dry weight of spleen (on a basis of 23 per cent of solids in fresh spleen). In Experiments 1 and 2 above only the cholesterol content of the splenic tissue is given (that obtained by deducting the cholesterol added). 23 Work to be published in the Journal of Experimental Medicine upon correlation of chemical and histological changes.

Harry J. Corper 45 Normal (Spleen B. Ref. 3)..; Experiment 2 (above)./ Experiment 1 (above)..i *Six days autolysis (spleenh, Ref. 3) *Two days i7~vivo autolysis (Spleen I, 0.00 ~ 53 hours 92 hours 6 days 0.0150 0.0162 0.0169 0.0216 Ref. 2)... ~ 48 hours in viva I 0.0217 *These two spleens were analysed at the mxne time; the normal spleens above being analysed at an earlier period. The last two cholesterol figures though differing from the first three by about 20 per cent are still within the error limit of the method. - In conclusion we can say then that within the limit of error of the Ritter method for cholesterol, this constituent of the tissues does not markedly change in amount during autolysis. GENERAL SUMMARY. 1. A source of error was found in the quantitative estimation of cholesterol by the Ritter method, in the fact that the presence of an excess of sodium alcoholate over that necessary for the saponification of the fats and esters, prevents a complete extraction of the cholesterol from the salt mixture by means of ether. 2. This error may vary from 5 per cent to 20 per cent in the case of a normal tissue when there is an excess of from 1 cc. to 3 cc. of a 5 per cent scidium alcoholate solutidn used in the saponification of 1.5 grams of the alcohol-ether extract. 3. The Ritter method for the quantitative determination of cholesterol in tissues should be used only with certain restrictions and precautions in mind. 4. No marked change was found in the amount of cholesterol present in the dog spleen after in vitro and in viva autolysis of short duration. 5. The steer spleen contains about 0.4 per cent of its moist weight as cholesterol.

ERRORS IN THE QUANTITATIVE DETERMINATION OF CHOLESTEROL BY RITTER'S METHOD: THE INFLUENCE OF AUTOLYSIS UPON CHOLESTEROL Harry J. Corper J. Biol. Chem. 1912, 11:37-45. Access the most updated version of this article at http://www.jbc.org/content/11/1/37.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/11/1/37.citation.full.html #ref-list-1