560 



Miss Eraser and Mr. Gardner. Origin and [June 17, 



dissolves at 18° only 0'014 gramme, at 78° about 0'16 gramme ; 100 c.c. of 

 50-per-cent. alcohol, at a boiling temperature, dissolves 0'03 gramme. It is 

 readily soluble in pyridin and slightly so in methyl alcohol. On these 

 properties Windaus based an accurate method* for estimating cholesterol, 

 which he applied to the determination of the amount of free and combined 

 ■cholesterol in kidneys. The mode of procedure we adopted was worked 

 out before the appearance of Windaus' later paper, and differs in detail 

 from that recommended by him. Our object was to devise a plan suitable 

 for the estimation of small quantities of cholesterol in material limited in 

 quantity, and to ascertain, as far as possible, the limits of accuracy attainable 

 lunder these conditions. 



Method. — The blood was mixed with plaster of Paris and the dry product 

 finely powdered and submitted to prolonged extraction with ether. For the 

 estimation of free cholesterol the ethereal extract was evaporated to dryness, 

 the residue weighed and taken up with 95-per-cent. alcohol. Excess of 

 digitonin in 95-per-cent. alcohol was then added, and the mixture, after 

 standing some time, was evaporated 'to diyness in a vacuum desiccator. 

 The precipitate was then washed by decantation with ether into a previously 

 weighed filter paper or Gooch crucible, until the ethereal washings gave 

 aio residue on evaporation. The excess of digitonin was then washed 

 away by warm water. In most cases the filtration proved tedious, and it 

 •was found more satisfactory, on the whole, to use a tared filter paper, care 

 being taken to subject the tare to exactly the same treatment as the filter 

 paper which received the precipitate. The washing with water was continued 

 until there was no residue on evaporation. The precipitate was then dried 

 in an air-oven at 110° C. and weighed, both drying and weighing being 

 •carried out in stoppered glass bottles, as the compound is somewhat 

 hygroscopic. In order to estimate the cholesterol present in the form of 

 ■esters, the ethereal washings containing the fat and cholesterol esters may 

 be saponified with excess of sodium ethylate, the unsaponifiable matter 

 dissolved in alcohol, and precipitated with digitonin as above. It is preferable, 

 however, should the amount of material available permit, to take a separate 

 quantity of material, and after extracting with ether, saponify the residue, 

 and, estimating the total free and combined cliolesterol present, obtain the 

 amount of ester cholesterol l)y dinerence. 



In order to test the eflficiency of the method, the following experiments were 

 carried out: — 



I. Experiments using small ([uantities of i)ure cholesterol, which were 



* VViiidauH, ' ZcitHclii ift fiir phyHiol. C'heni.,' 1!)10, vol. 65, j). 110. 



