2Q6 PHYSIOLOGICAL CHEMISTRY 



fatty acid solution 1 from a pipette with stirring to 50 c.c. of distilled water. To 

 the standard and to the test solutions are added simultaneously from pipettes and 

 with stirring 10 c.c. portions of dilute (1:3) hydrochloric acid and the solutions 

 allowed to stand for five minutes, after which they are transferred to the comparison 

 tubes of the nephelometer (see Fig. 83, p. 291). Several readings should be taken 

 and averaged. The standard tube should always be on the same side. See dis- 

 cussion of nephelometer (page 290) for details as to reading. The results repre- 

 sent the amount of total fat (fatty acids and cholesterol) in the blood, expressed 

 as oleic acid. The fat of the corpuscles is not completely extracted, and it should 

 be borne in mind that other lipoids as cholesterol are included in the results. 

 Cholesterol may be determined separately and subtracted from the result for total 

 fat. It may also be determined in a part of the blood extract as prepared above 

 by a modified Autenrieth-Funk procedure. 2 Methods have also been devised for 

 the determination of the phosphatides of blood. 3 



Instruments Used in the Examination of the Blood 



Spectroscope. 4 Either the angular -vision spectroscope (Figs. 87 

 and 88) or the direct-vision spectroscope (Fig. 86) may be used in 

 making the spectroscopic examination of the blood. For a complete 

 description of these instruments the student is referred to any standard 

 text-book of physics. 



FIG. 86. DIRECT-VISION SPECTROSCOPE. 



1. Oxyhemoglobin. Examine dilute (1:50) defibrinated blood spectro- 

 scopically. Note the broad absorption band between D and E. Continue the 

 dilution until this single broad band gives place to two narrow bands, the one 

 nearer the D line being the narrower. These are the typical absorption bands 

 of oxyhemoglobin obtained from dilute solutions of blood. Now dilute the blood 

 very freely and note that the bands gradually become more narrow and, if the 

 dilution is sufficiently great, they finally entirely disappear. 



2. Hemoglobin (so-called Reduced Hemoglobin). To blood which has been 

 diluted sufficiently to show well-defined oxyhemoglobin absorption bands add a 

 small amount of Stokes' reagent. 5 The blood immediately changes in color from 



1 The standard solution used is an alcohol-ether solution of pure oleic acid of which 

 5 c.c. contain about 2 mg. of the acid. The alcohol and ether used for the standard are 

 freshly redistilled absolute alcohol and pure dry ether. 



2 Bloor: Jour. Biol. Chem., 23, 317, 1915. 



3 Greenwald: Jour. Biol. Chem., 21, 29, 1915. 



Bloor: Jour. Biol. Chem., 22, 133, 1915, 23, 317, 1915. 



Kober and Egerer: /. Am. Chem. Soc., 37, 2373, 1915. 



Taylor and Miller: Jour. Biol. Chem., 18, 215, 1914. 



For other nephelometric methods see Chapters XVII and XXVI. 



4 For Absorption Spectra see Plates I and II. 



6 Stokes' reagent is a solution containing 2 per cent ferrous sulphate and 3 per cent tar- 

 taric acid. When needed for use a small amount should be placed in a test-tube and ammo- 

 nium hydroxide added until the precipitate which forms on the first addition of the hydrox- 

 ide has entirely dissolved. This produces ammonium ferrotartrate which is a reducing agent. 



