ANALYSIS OF TISSUES 585 



t>. CARBOHYDRATES. 



The carbohydrates which are present in the tissues used as foodstuffs consist 

 mainly of starch. Small quantities of glucose may also be present. Biscuits, 

 cakes, etc., will contain cane sugar in addition. 



Fresh animal tissues contain glycogen. 



Estimation of Starch. 



The simplest method of estimating starch is a modified form of the Pfliiger 

 method for estimating glycogen in animal tissues ; as adopted by Armstrong 

 it is as follows : 



10 gm. of the substance are boiled for half an hour with 80 c.c. of 5 per 

 cent, potassium hydrate under a reflux condenser. The mixture must be well 

 shaken at first as frothing occurs ; the frothing can be lessened by adding a few 

 drops of amyl alcohol. The liquid, generally brown in colour, is cooled and 

 poured into 200 c.c. of 80 per cent, alcohol. The starch is precipitated ; it is 

 stirred to cause it to agglomerate and allowed to settle ; it is filtered off, washed 

 with dilute alcohol and put into a 300 c.c. flask, in which it is hydrolysed by boil- 

 ing with 200 c.c. of 7-5 percent, (by volume) hydrochloric acid for 2-5-3 

 hours. The solution is neutralised, made up to 250 c.c. and the glucose 

 estimated by any of the methods for estimating glucose (p. 216). 



Estimation of Cane Sugar and Glucose. 



10 gm. of the powdered material are covered and stirred up with "4 per 

 cent, hydrochloric acid. The acid is decanted through a filter paper into a 

 measuring flask of 250 or 500 c.c. capacity. The material is again covered with 

 acid and the acid decanted. The extraction is continued in this way some six or 

 seven times until a test portion shows the absence of glucose and cane sugar. 

 The solution is diluted to the mark. 



In one or more aliquot portions the glucose is estimated and in one or 

 more portions the cane sugar is estimated according to the methods given on 

 p. 235. 



Estimation of Glycogen (Pfliiger's method). 



The amount of glycogen in animal tissues varies considerably so that small 

 or large quantities of material must be taken accordingly. 



The shorter method of Pfliiger 1 gives good results : 



100 gm. of the minced tissue are put into 100 c.c. of boiling 60 per cent, 

 potassium hydroxide and heated for about two hours until the tissue has com- 

 pletely dissolved (excepting connective tissue . On cooling, 200 c.c. of watei 

 are added and thoroughly mixed with the solution. The glycogen is pre- 

 cipitated by adding 400 c.c. of 96 per cent, alcohol. The precipitate is 

 allowed to settle and the liquid is decanted through a 15 cm. filter paper. 

 The precipitate is washed once with a mixture of i volume of 15 per cent, 

 potassium hydroxide and 2 volumes of 96 per cent, alcohol, finally with 66 

 per cent, alcohol. It is dissolved in boiling water ; the filter paper and in- 

 soluble residue are also boiled out with water. If there be a large separation 

 of protein with the glycogen, it is boiled out twice. The aqueous solutions 

 are neutralised. Concentrated hydrochloric acid is added to make 2 '2 per 

 cent, and the glycogen is hydrolysed by boiling for 3 hours. The solution is 

 neutralised and filtered and the glucose is estimated by polarimeter or by 

 reduction. 



Glycogen = glucose x -927. 



1 Pfluger's Arch., 103, 169. 



