328 PRACTICAL PHYSIOLOGY 



Windaus that cholesterol, but not cholesterol esters, readily combines 

 with digitonin to form a very insoluble compound, methods have been 

 introduced for the quantitative estimation of cholesterol by gravimetric 

 methods. One of the simplest is that used by Gardner. 



Grind up the tissue with sand and a sufficient amount of plaster of 

 Paris to make it set. When the mass is dry it is finely powdered and 

 thoroughly extracted with ether in a Soxhlet apparatus. (Extraction 

 requires several days at least. ) The ethereal extract is then evaporated 

 to dryness, the residue weighed and taken up in 95 per cent, alcohol. 

 To this solution is added an excess of digitonin in 95 per cent, alcohol 

 and the mixture after standing for some time is evaporated to dryness 

 in a vacuum desiccator. The precipitate is next washed, by decantation 

 with ether, into a previously weighed filter paper or Gooch crucible 

 until the washings give no residue on evaporation. The excess of 

 digitonin is next removed by washing with warm water until the 

 washings give no precipitate on evaporation. The precipitate is 

 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. 



If it be desired also to determine the amount of cholesterol present in 

 ester form the ethereal washings from the above estimation are 

 saponified with excess of sodium ethylate, the unsaponifiable matter 

 dissolved in alcohol, precipitated with digitonin and treated as above. 

 Or preferably, if material permit, it is better to do two estimations, (a) 

 total free and (6) total free and combined cholesterol present, then (6) 

 minus (a) will give combined cholesterol. 



Cholesterol has also been estimated by colorimetric methods (see 

 Blood, p. 318). 



Preparation of Glucosamine from Chitin. Soak lobster or crab shells 

 in dilute (2 per cent.) hydrochloric acid until all calcium carbonate is 

 removed and the material is soft and pliable. Wash well with water, 

 then cut into small pieces, place them in a round-bottomed flask, add 

 concentrated hydrochloric acid and boil gently under a reflux condenser 

 for four hours. The resultant solution is then evaporated on the 

 waterbath in an evaporating basin until crystallisation commences ; 

 allow to cool. Filter off the crystal mass, wash carefully with a small 

 quantity of cold water and dilute alcohol. The crystals are again 

 dissolved in water and the solution again evaporated until commencing 

 crystallisation. Filter off the crystals : to free them from any in- 

 organic material dissolve the mass in 80 per cent, alcohol, filter, and 

 again evaporate to crystallisation. The crystal mass of glucosamine 

 hydrochloride which is formed should be almost pure. Glucosamine 

 forms an osazone identical with glucosazone. Its solutions reduce 

 Fehling's solution. 



CHAPTER XXIV 

 FOODSTUFFS AND METABOLISM 



Milk. Milk contains proteins, fats, carbohydrates, salts and water. 

 The fat is suspended in the form of a fine emulsion. The proportion of 

 these bodies varies in the milk of different animals. Naturally that 

 provided by the animal is the best for its own species. 



