were in good agreement whether obtained 

 by the oxidative procedure or by multiply- 

 ing the phosphorus present by the factor 25. 

 It was found that the temperature of evapora- 

 tion of the alcohol -ether extract affects the 

 solubility of the phospholipids in petroleum 

 ether; therefore the use of a vacuum and 

 temperature of less than 50° is recommend- 

 ed for the evaporation. CHCI3 dissolves the 

 phospholipid-MgCl2 precipitate as well as 

 moist ether, giving more consistent results 

 and requiring less time for solution. Alco- 

 hol-ether extracts of plasma, when stored 

 for several months gave ratios of lipid phos- 

 phorus, phospholipid, and phospholipid fatty 

 acid no lower than extracts used a few days 

 after preparation . See also: Boyd ( Journal 

 of Biological Chemistry , 121 : 485, 1937) 

 and Halliday (Journal of Nutrition, 16 : 285, 

 1958). 



Engel. R. W. 



1942. Modified methods for the chemical 



and biological determination of choline . 



Journal of Biological Chemistry, 144: 



701-710. 

 Choline was extracted from biological 

 material by continuous extraction with meth- 

 anol, which is claimed to be the most ef- 

 fective solvent (obtained recoveries within 

 1.5% of theoretical) . (Brante (Acta physio - 

 logjca Sca ndinavia,^- supplement 63, 1949) 

 states that ethanol-ether (3:1) is just as ef- 

 fective as methanol). The extract was con- 

 centrated and hydrolyzed with Ba(OH)p at 

 100° C. (100° was used since the tempera-* 

 ture of 80° suggested by Jacobi ( Journal of 

 Biological Chemistry, 138: 571, 1941) fail- 

 ed to give consistent results.) Choline was 

 determined as the reineckate by colorimetry. 

 The incidence of kidney hemmorhage in rats 

 was also used as an indication of the quantity 

 of choline and choline-like materials in bio- 

 logical test diets, and showed sensitivity to 

 a variation of 10% of the materials . 



Engel, R. W., W. D.Salmon, and 

 C.J. Ackerman 



1954. Chemical estimation of choline. 

 In Methods of Biochemical Analysis , 

 D. Click, Editor, New York, Inter- 

 science Publishing, Inc., Vol. 1, 



pp. 265-286. 

 A study and discussion of various meth- 

 ods for the estimation of choline in tissues. 



Entenman, C, A. Tkurog, and I. L. Chaikoff 



1944. The determination of choline in 

 phospholipids . Journal of Biological 

 Chemistry, 155: 13-18. 



The phospholipids were hydrolyzed with 

 Ba(OH)2 and the choline was precipitated 

 . as the reineckate and read colorimetrically. 

 Accuracy j- 3% using choline chloride. 



1 .2 N HCl was used as solvent for ammo- 

 nium reineckate, making it possible to use 

 a more concentrated reineckate solution. 

 Precipitation is complete in 30 minutes. 



Entenman, C . and I . L . Chaikoff 



1945. On the determination of choline in 

 the liver and plasma of the dog. Jour- 



A nal of Biological Chemistry, 160 : 377- 



385. 

 Various extracts and fractions of plasma 

 and liver were analyzed for choline by the 

 methods of Click (Journal of Biological 

 Chemistry , 156 : 643, 1944) and of Enten- 

 man, et al ( Journal of Biological Chemistry , 

 155 : 13, 1944). Identical choline values 

 were found for the phospholipid fraction 1- 

 solated from either liver or plasma when 

 the precipitations of choline reineckate were 

 carried out in the presence of 1 .2 N HCI 

 (Entenman, et al) or at a pH between 8 and 

 9 (Click). Both methods gave values in 

 agreement on analyses of both alcohol - 

 ether and methanol extracts of plasma, but 

 Click's method gave lower values for an 

 alcohol -ether extract of liver. 



Erickson, B. N., I. Avrin, D. M. Teague, and 

 H. H. WiUiams 



1940. Micromethods for the determination 

 of sphingomyelin and choline. Applica- 

 tions for the estimation of the phospho- 

 lipid partition (sphingomyelin, lecithin, 

 and cephalin) in blood and urine . Jour- 

 nal of Biological Chemistry, 135: 671- 

 684. 

 A procedure is described in which sphingo- 

 myelin is precipitated as the reineckate and 

 calculated from the phosphorus content of 

 the reineckate. Choline is determined, after 



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