ADVANCED CHEMICAL PHYSIOLOGY 297 



are recovered. When the quantity of lactic acid is considerable, 

 however, the loss is negligible. 



In order to apply the method to blood the following preliminary 

 procedure is necessary. The blood, of which 20 c.c. is usually quite 

 sufficient, is diluted about five times, heated to boiling in order to 

 coagulate the proteins, and filtered. The coagulum is very thoroughly 

 washed with boiling, faintly acidulated water. The total liquid thus 

 obtained is rendered alkaline with sodium carbonate, evaporated and 

 employed for the determination. 



Estimation of Urochrome. Add about 20 gms. finely powdered 

 ammonium sulphate to 25 c.c. urine. Actively stir the mixture with 

 a glass rod so that most of the ammonium sulphate is dissolved. Allow 

 to stand for at least thirty minutes in order that other pigments present 

 may precipitate, then fill up to 50 c.c. with hot saturated ammonium 

 sulphate solution and filter at once. 10 c.c. of the filtrate is placed 

 in one of the cups of a Duboscq colorimeter and compared with a 

 standard solution of " Echtgelb " (Muller, Leipzig) containing 0-Olg. 

 Echtgelb in 2 litres of water. 



Urobilin. -Urobilin is a derivative of haemoglobin and is probably 

 identical with the faecal pigment stercobilin. It readily combines with 

 alkalis to form salts which are easily soluble in water. Urobilin 

 shows a well-marked absorption band in the green near F. If urine 

 is first rendered acid with a mineral acid or if a drop or two of tincture 

 of iodine be added the chances of getting the spectrum in urine are 

 much increased. Its presence can also be detected in urine by its 

 fluorescence, particularly in the presence of zinc salts. 



The Schlesinger Test. Take 10-15 c.c. urine in a wide test tube 

 and add to it an equal volume of well-shaken zinc acetate solution, 1 

 mix well and allow to stand for twelve to twenty-four hours in order 

 to allow the precipitate to settle. The clear supernatant fluid of 

 urobilin, if present, will show a beautiful green fluorescence. A better 

 result is obtained by filtering off the precipitate and examining the 

 column of fluid for fluorescence from above. If the column appears 

 colourless no urobilin is present, or only a minute amount, but if it 

 appear yellowish red or rose it may be regarded as pathological. 



A more refined method of carrying out this test is to add 3-4 drops 

 glacial acetic acid to 50 c.c. urine, rendering the reaction definitely 

 acid. Then drop by drop, shaking well, 1 per cent, tincture of iodine 

 (1 drop to each 2 c.c. of urine). Add 5 c.c. thymol chloroform and 

 shake thoroughly. The chloroform extract is mixed with an equal 

 volume of an alcoholic zinc acetate solution (alcohol 93 per cent. 

 500 c.c., zinc acetate 3 gms., glacial acetic acid 2 c.c. ; filter before 

 use), shake and filter. The presence of a beautiful green fluorescence 

 is indicative of the presence of urobilin. 



Preparation of Urobilin (Garrod and Hopkins). Saturate the urine 

 with ammonium chloride in order to remove uric acid, filter, acidify 

 the filtrate with sulphuric acid, saturate with ammonium sulphate. 

 Extract this mixture in a large separating funnel with an equal volume 

 of a mixture of 1 part chloroform and 2 parts ether. The chloroform 

 ether extracts the urobilin. The extract is then shaken with faintly 

 alkaline water which takes up the urobilin. In order to purify the 



1 10 gms. of zinc acetate are added to 100 c.c. alcohol. Most of the acetate 

 remains undissolved, hence the reagent must be well shaken before use. 



