604 
Vol. XXIX, No. 12 
Journal oj Agricultural Research 
a. m. and at 3.15 to 3.45 p. m.); and, 
so far as possible, the milking was done 
by the same man throughout each 
experiment. The blood samples were 
taken at 8.30 to 9 a. m. 
In all the experiments except Nos. I 
and II the Ca, P, and Na contents of 
the rations were kept constant by 
adding varying amounts of ground 
limestone, Na 2 HP0 4 . 12H 2 0,and NaCl, 
as shown in Table II. The calcula¬ 
tions of the amounts of these salts to 
be added were based on determinations 
found in the literature or made at 
various times in this laboratory. The 
cows also had access to NaCl when 
turned out daily in a bare lot. 
ANALYTICAL METHODS 
The samples of blood in Experiments 
I, II, III, V, and VIII were 450 to 1,000 
c. c. They were collected in bottles 
containing 0.1 gm. of sodium oxalate 
er 100 c. c. of sample. In Experiments 
V, VI, and VII the samples of blood 
were larger; and 0.7 c. c. of a solution 
of sodium citrate (38 gm. per 100 c. c. 
of solution) was used per 100 c. c. blood. 
The plasma was immediately separated 
by centrifuging 20 minutes at 3,600 
revolutions per minute. The analyti¬ 
cal samples for the blood and plasma 
determinations were taken out as 
quickly as possible. 
The lipoid phosphorus of the blood 
plasma was calculated as the difference 
between the total and the inorganic 
phosphorus in the plasma as determined 
according to the methods described by 
Meigs, Blatherwick, and Cary (11). 
The amino acid N of the blood and 
plasma was determined as previously 
described ( 3 ) except that, beginning 
with the sample of July 22, Experiment 
I, the urease was reduced to one-tenth 
that previously used. The determina¬ 
tions on the Van Slyke apparatus were 
made at the same room temperature 
throughout each experiment. The re¬ 
ducing sugar of the blood and plasma 
was determined by the revised method 
of Folir. and Wu*(£). The blood cor¬ 
puscle volume was measured off on a 
marked centrifuge tube. 
The p-dimethylamidobenzaldehyde 
reagent of Herzfeld (8) was used in 
the determination of the tryptophane 
of the blood and blood plasma. The 
protein was precipitated either as in 
the amino N determination (cow 246), 
or by coagulation with acetic acid as 
in the amino N determination and 
subsequent precipitation with alde¬ 
hyde-free alcohol 3 (cow 423). The 
tryptophane in the concentrated pro¬ 
tein-free extracts was precipitated in 
the presence of 7 per cent (by volume) 
of sulphuric acid and 20 per cent of 
mercuric sulphate. The mercury- 
tryptophane precipitate was redissolved 
in H 2 0 and 5 per cent NaCN (total 
volume = 5 c. c.). Then 2 c. c. of the 
reagent and 23 c. c. of hydrochloric 
acid were added. With cow 246 
hydrochloric acid that had been sat¬ 
urated at 0° C. was used. With cow 
423 ordinary hydrochloric acid was used 
and then HC1 gas was run in slowly for 
10 minutes, while cooling in tap water. 
They were then heated at about 60° C. 
in a water bath until the color was fully 
developed (30 minutes). They were 
compared with tryptophane standards 
similarly treated with the Herzfeld 
reagent, HC1, etc. Generally, three 
or more standards were run, because, 
although they usually agreed well, it 
was found that sometimes they did 
not. This method of determining the 
protein-free tryptophane is not entirely 
satisfactory; the colors do not match 
well. In Experiments IV and VI the 
unknowns and standards were diluted 
1:2 with water before reading. The 
writers believe that the data for free 
tryptophane obtained by them are 
sufficiently reliable to warrant publica¬ 
tion and the conclusions that they have 
drawn from them. Further work is in 
progress upon methods of determining 
free tryptophane in blood and plasma. 
The results of these investigations will 
be published later. 
The milk was preserved by adding to 
each 100 c. c. either 10 drops of chloro¬ 
form (Experiment I), 2 c. c. of formalin 
(Experiments II, III, V, and VII), or 
2 c. c. of a mixture of 50 gm. of phenol 
and 10 c. c. of 95 per cent alcohol 
(Experiments IV, VI, and VII). The 
nitrogen in the milk was determined by 
the Kjeldahl-Gunning-Arnold (1) 
method, the fat by the ordinary Bab¬ 
cock method, and the lactose by means 
of a polariscope. The proteins in the 
lactose determinations were precipi¬ 
tated by acid mercuric nitrate accord¬ 
ing to the “Methods of Analysis” of 
the Association of Official Agricultural 
Chemists (1). 
The analytical data shown in the 
tables are generally averages of two or 
more determinations except where 
lactose was used, in which case only 
single determinations were made. The 
tryptophane determinations in Experi¬ 
ment VIII were not run in duplicate. 
The changes made in the rations and 
the effects thereby brought about on 
the composition of the blood and milk 
and on the quantity of milk yielded 
are given in detail in Tables I to X. 
3 Refluxed and distilled off from KOH. 
