mination of the a-amino nitrogen of the amino acids. This method

19 Pages·2003·0.94 MB·English

AMINO ACID NITROGEN IN BLOOD AND ITS DETER-
MINATION
BY IRVIN S. DANIELSON
(From the Biochemical Laboratory of Harvard Medical School, Boston)
(Received for publication, May 13, 1933)
In 1922 Folin (1) proposed a method for the calorimetric deter-
mination of the a-amino nitrogen of the amino acids. This
method is based on the production of a yellow-orange color by
the reaction of the amino acids with P-naphthoquinonesulfonic
acid in alkaline solution. The intensity of the color produced is
compared with the color produced by a known amount of glycine
treated in the same manner. This method was developed mainly
for the determination of a-amino acid nitrogen in blood filtrates
and urine but is applicable to other fluids containing amino acids.
The method has been widely used in the study of amino acids
occurring in the blood stream. In general, satisfactory results
have been obtained. During the course of a study on the concen-
tration of a-amino acid nitrogen in whole blood based on the analy-
sis of unlaked blood filtrates, it was noticed that the range of t’rue
proportionality over which the color comparisons could be made
was relatively small. Upon investigation it was found that this
limitation was entirely due to the presence of a strong blank caused
by the incomplete bleaching of the excess quinone reagent.
Edgar (2) found that by adding varying amounts of alkali to a
series of determinations on the same sample of filtrate a variety
of results was obtained. Re and Potick (3) reported failure in
obtaining satisfactory results by this method. The results of
Edgar are easily explained by the variable blank present which
thus gave an apparent increase in the amino nitrogen content of the
samples analyzed. Undoubtedly the presence of this strong blank
was a factor in the unsatisfactory results reported by Re and
Potick. However, their results were at such variance with all
other published results that some other serious error must have
been included in their work also.
505
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506 Amino Acid Nitrogen in Blood
It is the purpose of this paper (1) to describe a modification of
the original Folin method; this modification will give determina-
tions containing no blank, thereby increasing the range of true
proportionality; (2) to present evidence showing that the tungstic
acid filtrates prepared from unlaked blood give true values for the
free amino acid nitrogen in the blood stream and that the values
found in laked blood filtrates are much too high; and (3) to record
amino acid nitrogen values found in the blood of normal men and
animals when this analysis is based on the unlaked blood filtrate.
Description of ModiJication Proposed in Amino Acid Nitrogen
Method
The principle of the modified procedure is the same as that of
the original method, but each solution except the P-naphtho-
quinone sulfonic acid has been changed in detail. These changes
will be described and the reasonsf or each will be briefly stated.
The solutions used were the following.
Standard Amino Acid Solution-Two stock solutions are pre-
pared; one contains 0.1 mg. of amino nitrogen per cc. as glycine
dissolved in 0.07 N HCl plus 0.2 per cent sodium benzoate, and the
other contains 0.1 mg. of amino nitrogen per cc. as glutamic acid
in 0.07 N HCI plus 0.2 per cent sodium benaoate. The sodium
benzoste is used as a preservative. Standards used in the analysis
of blood filtrates are prepared from these by mixing equal volumes
of the two stock solutions and diluting with 0.07 N HCl containing
0.2 per cent sodium benzoate to a concentration of 0.03 mg. and
0.05 mg. of amino nitrogen per cc. A 0.03 mg. standard is pre-
pared by adding 15 cc. each of the glycine stock solution and the
glutamic acid stock solution to a 100 cc. volumetric flask. The
contents are made up to a volume of 100 cc. with 0.07 N HCI con-
taining 0.2 per cent sodium benzoate. The 0.03 mg. standard
is used for the analysis of filtrates prepared from normal human
unlaked blood. The amino acid concentration in most animal
bloods is appreciably greater than that of human blood, therefore
the strength of the standard used for such comparisons should be in-
creased accordingly. It is not permissible to use 2 cc. of a weaker
standard to prepare a standard of double strength due to the
fact that the standard is so prepared that 1 cc. contains about the
equivalent of acid found in 10 cc. of tungstic acid blood filtrate.
I. S. Danielson 507
If a stronger standard is required it is necessary to prepare it from
the stock solutions.
The use of both glycine and glutamic acid in the standard is
made necessary because the shades of color produced by the differ-
ent amino acids vary somewhat. The standard as recommended
produces a color that matches very nearly that produced in the
tungstic acid blood filtrates.
Borax Solution-. As a source of alkali a 1.5 per cent solution of
borax is used. Borax is used instead of the sodium carbonate
solution for with its use less blank remains and the results are
more satisfactory under the conditions recommended. A definite
volume (2 cc.) of borax is added to each determination.
Bleaching Reagents-Two solutions are needed for the bleaching
of the excess /%naphthoquinonesulfonic acid reagent: (1) a 0.1 M
solution of sodium thiosulfate which need not be standardized,
and (2) an acid formaldehyde solution prepared by mixing 3 vol-
umes of 1.5 N HCl and 1 volume of glacial acetic acid with 4 vol-
umes of 0.15 M formaldehyde. The 0.15 M formaldehyde solution
may be prepared accurately enough by diluting 11.3 cc. of ordinary
40 per cent formaldehyde to 1000 cc.
Sodium thiosulfate in acid solution has the property of destroy-
ing the color of p-naphthoquinonesulfonic acid. If acetic acid is
used in making this solution acid the bleaching of the quinone is
far from complete but if a strong acid like hydrochloric acid is used
the color is bleached completely. However, in the presence of a
strong acid, sodium thiosulfate immediately decomposes with the
liberation of sulfur. The addition of an amount of formaldehyde
equivalent to the thiosulfate present will delay this decomposition
for many hours. In the solutions as recommended the thiosulfate
is present in a slight excess over the formaldehyde, i.e. 2 cc. of
0.1 M thiosulfate and 2 cc. of 0.075 M formaldehyde. Under these
conditions the bleaching is more prompt and complete, and the
solution remains perfectly clear for several hours.
The addition of acetic acid to the acid formaldehyde is made
necessary because an amino acid determination made on a solution
containing tryptophane will become cloudy in the absence of
acetic acid. Tryptophane is the only amino acid we have studied
that behaves in this manner. Determinations on unlaked blood
filtrates develop a very slight cloud if acetic acid is not included in
508 Amino Acid Nitrogen in Blood
the acid formaldehyde solution. This cloud is presumably due to
tryptophane present in the filtrate.
Sulfate-Tungstate Solution for Addition to Standard-This solu-
tion contains 15.0 gm. of NazS04 (anhydrous) and 1.5 gm. of
Na2W04.2Hz0 per 1125 cc. of solution.
The unlaked blood filtrate contains about 1.5 per cent Na#OA
and about 0.15 per cent Na2W04.2Hz0 which remains from the
solutions used in the preparation of the filtrate. The presence of
these salts in the above concentrations has very little or no effect
on the amount of color developed in the determination but sodium
tungstate in particular and sodium sulfate to a much smaller
degree alter the shade of color produced, making exact color
comparisons difficult. The presence of sodium tungstate produces
a disturbing greenish shade. 9 cc. of the above solution are, there-
fore, added to the standard to balance this effect and to bring the
volume of the standard up to that of the unknown.
If sodium sulfate is present in concentrations of 3 per cent or
more, or sodium tungstate in concentrations of 0.6 per cent or more,
an error of 2 per cent or more, depending on the concentrations,
will be introduced.
/3-NaphthoquinonesuiJonic Acid Solution-We are still using this
solution as originally recommended, that is, a freshly dissolved 0.5
per cent solution of @naphthoquinonesulfonic acid in water.
The solid reagent is prepared according to the directions given by
Folin.
Description of Determination
The amino acid nitrogen content of a blood filtrate may be
determined on either 5 cc. or 10 cc. of filtrate. When 10 cc. of
filtrate are used the procedure is as follows:
Transfer 10 cc. of the filtrate into a test-tube graduated at the
25 cc. mark. To this add 2 cc. of 1.5 per cent borax solution and
2 cc. of a freshly prepared 0.5 per cent ,!%naphthoquinonesulfonic
acid solution and mix thoroughly. The standard is prepared by
introducing 1 cc. of the standard solution of desired strength into
a test-tube similar to that used for the filtrate. To the standard
that is to be used in the comparison of an unlaked blood filtrate or
plasma filtrate add 9 cc. of the sulfate-tungstate solution prepared
for this purpose. If the filtrate to be analyzed contains no tungstic
I. S. Danielson 509
acid and very little sodium sulfate (laked blood filtrate), add 9 cc.
of wat,er. Then add to the standard 2 cc. of the 1.5 per cent borax
solution and 2 cc. of the 0.5 per cent P-naphthoquinonesulfonic
acid solution and mix well. Both standard and unknown are
then set in a dark closet for 18 to 24 hours. After this period of
standing add 2 cc. of the acid formaldehyde solution and 2 cc. of
the 0.1 M sodium thiosulfate solution. Dilute the contents of each
tube to a volume of 25 cc. with distilled water and then mix thor-
oughly. After standing for 4 to 5 minut’es to allow for the complete
bleaching of the excess quinone reagent, the unknown is compared
wit,h the standard by the use of a calorimeter.
If 5 cc. of the blood filtrate are used for the determination, add
1 cc. of the borax solution and 1 cc. of the 0.5 per cent quinone
solution t,o t.he filtrate. The standard is prepared as described
above. After the 18 to 24 hour period of standing add 1 cc. of each
t,he acid formaldehyde and the thiosulfate solutions to the un-
known, dilute to 15 cc., and mix. To the standard add 2 cc.
portions of the acid formaldehyde and thiosulfate solutions, dilute
t,o 30 cc., and mix. Color comparisons are made as directed above.
The calculation in either case is made by the use of the following
formula,
20/R X 0.03 X 100 = mg. per cent or 20/R X 3 = mg. per cent
when the calorimeter setting for the standard is 20. R is the
reading of the unknown and 0.03 mg. is the concentration of t,he
amino nitrogen in the standard.
When the amino acid nitrogen of fluids other than blood filtrates
is to be det.ermined, the sample taken should contain between 0.03
and 0.15 mg. of amino nitrogen and should be neutralized with
sodium hydroxide or hydrochloric acid depending on whether the
sample is acidic or basic, phenolphthalein being used as indicat,or.
In order to balance the acidity of the unknown with the standard
add 1 cc. of 0.07 N HCl. The determination is then carried out
in the same manner as described for blood filtrates.
Amount of Color Developed by Various Amino Acids-The
amount of color developed by the various samples of pure amino
acids which we had on hand was compared with that produced by
two of the amino acids (i.e. glycine and glutamic acid) taken as
standards. In Table I are tabulated t,he results found. The
510 Amino Acid Nitrogen in Blood
shade of color developed by some of the amino acids is somewhat
djfferent from that produced by others. In the last column of
Table I an attempt is made to indicate how these shades appear in
the calorimeter when compared with the standard. In comparing
the color produced by two amino acids, which show a difference in
shade, the criterion for the comparison should be the matching of
TABLE I
Values Obtained from Different Amino Acids Compared with Glycine and
with Glutamic Acid As Standards
N&-N
Ixperi. Aver- Shade compared with
Standard merit Amino acid age standard
NO. Added Found error
j mg. 1 VW. ( ,“,“,‘t
Glycine 1 Alanine 0.075 0.0754 0.5 Shades good
2 Cystine 0.075 0.073 2.7 Greenish yellow
3 Lysine 0.03750.0355 5.3 Much yellower
4 Tyrosine 0.075 0.0741 1.2 Yellower
‘I
5 Histidine 0.075 0.0746 0.5
6 Aspartic acid 0.075 0.0743 0.9: “
“
7 Glutamic “ 0.075 0.0748 0.2(
8 Leucine 0.075 0.0748 0.2( Shades good
9 Tryptophane 0.075 0.075 0.0 Little grayish
10 Serine 0.07 0.07 0.0 Shades good
Glutamic Alanine 0.075 0.0765 2.0 Purplish tan tinge
acid Cystine 0.075 0.0721 3.8 Gray tinge
Lysine 0.03750.0365 2.7 Much yellower
Tyrosine 0.075 0.0748 0.21 Little “
Histidine 0.075 0.0761 1.4 Shades good
‘I “
Aspartic acid 0.075 0.0745 0.6
Glycine 0.07 0.0706 0.81 Purplish tan tinge
Leucine 0.075 0.076 1.3 Shades good
the amount of yellow color in each and not the intensity of light
allowed to pass through the solutions.
While this difference in the shade of color developed by two
different amino acids may be quite disturbing when viewed in a
calorimeter, it should not be stressed unduly for it is not great.
No difference in shade can be noted when two determinations in
test-tubes are compared with the naked eye. However, it, is
I. S. Danielson 511
well in analyzing for amino acid nitrogen to choose a standard
which matches exactly with the unknown. This will make color
comparisons easier and the error introduced in judging the exact
comparison by different individuals will be reduced. It is because
the shade of color developed by a blood filtrate is somewhat differ-
ent from glycine or any other amino acid easily obtained in the
pure state, that we are recommending the use of a standard con-
taining one-half of its nitrogen as glycine and one-half as glutamic
acid. This standard matches exactly a blood filtrate when used
as recommended.
A solution containing equal amounts of amino nitrogen of the
amino acids tested in Table I produces a shade of color that
matches very well with that produced by the standard recom-
mended for blood analysis and the proportionality is perfect.
In this connection it should be mentioned that ammonia reacts
with p-naphthoquinonesulfonic acid under the conditions recom-
mended and must therefore be removed from a solution to be
analyzed for amino acid nitrogen. The color produced by am-
monia has a decidedly purplish tint when compared with the color
produced by any of the amino acids. It is impossible to get a
good color comparison between ammonia and an amino acid but
ammonia seems to develop from 65 to 75 per cent of the color
developed by an equivalent amount of glycine.
Amino Acid Nitrogen in Whole Blood
In 1930, Folin (4) proposed the use of a filtrate prepared from
whole blood without hemolyzing the red corpuscles, as the basis for
the study of food products and waste products occurring in the
blood stream. The preparation of such a filtrate was accomplished
by simply diluting the blood with a slightly hypertonic solution
(1.5 per cent) of anhydrous sodium sulfate instead of distilled
water. The amino acid nitrogen found in a filtrate prepared in
this manner is much less than that found in a filtrate prepared by
laking the red corpuscles; i.e., the Folin-Wu tungstic acid filtrate.
In 1931, Simon (5) reported a study of the amino acid nitrogen
content of whole blood based on the analysis of both laked and
unlaked blood filtrates. He came to the conclusion that the value
found in the unlaked blood filtrate was too low and that for this
512 Amino Acid Nitrogen in Blood
determination, at least, the unlaked blood filtrate cannot be used
to give accurate results.
Simon presents two very plausible arguments along with experi-
mental data which apparent,ly seem to uphold his conclusions.
First, he makes the very reasonable suggestion that if in the prep-
aration of an unlaked blood filtrate the free amino acids are
freely diffusible to the extent characteristic of the blood, then on
the addition of amino acids to the whole blood it should be expected
that the increase in the amino nitrogen content of the red cor-
puscles, above that present before the addition of the extra amino
acid, should be the same whether this value is calculated from
data obtained from the analysis of filtrates prepared from laked
blood or from unlaked blood. His experimental data show that
this, apparently, is not the case. In the seven experiment’s
reported, he always found a greater increase in the corpuscle value
calculated on the laked blood analysis. He found that the
increase in the corpuscle values calculated on the unlaked blood
filtrate data was 40.2, 17.7, 74.3, 94.0, 32.7, 66.0, and 18.5 per cent
respectively of the increase in the value based on laked blood data.
In only one case did he find increases that were comparable; i.e.,
the increase found in the unlaked corpuscles was 94 per cent of
that found in the laked corpuscles.
Although Simon does not state exactly how much amino acid
was added in each experiment, it can be seen from his tables t’hat
complete recovery of the added amino acids was not obta*ined in
the majority of these experiments for in only one case is the increase
in the whole blood nearly the same in both the laked and unlaked
blood filtrates. This one case is the experiment in which nearly
equivalent increases in the amino nitrogen content of the cor-
puscles were found.
We have repeated these experiments, using our modified amino
acid nitrogen method, and in every case where the recovery of the
added amino acids to the whole blood was nearly complete we
found comparable increases in the corpuscle amino nitrogen by
calculating this value from data obtained from the analysis of
both laked and unlaked blood filtrates. The experimental data
will be presented later.
The second argument which Simon presents which tends to
show that the free amino acids of the corpuscles are not permitted
I. S. Danielson 513
to diffuse out of the cell into the sulfate-tungstate diluting fluid in
the preparation of an unlaked blood filtrate is simply that this
extra amino nitrogen can be washed out by resuspending the cor-
puscles in a second portion of sulfate-tungstate solution. Simon
found by this process of washing the cells, that the amino acid
nitrogen content of the t,wo washings was very nearly the same as
that found in a whole blood filtrate prepared by laking the red
corpuscles.
We have repeated these experiments also and in no case have
we found the amino acid nitrogen content of such washings to be
anywhere nearly as great as that found in a laked blood filtrate.
On the contrary the values found were very nearly the same as
t,hose of the unlaked blood filtrate. Our data are given in Table
III.
We can, therefore, see no justification in Simon’s conclusion
that in the preparation of the unlaked blood filtrate the free amino
acids do not diffuse out into the diluting fluid to the extent charac-
teristic of the blood. Further evidence based on the study of the
distribution of amino acids between the corpuscles and plasma
is presented supporting this view.
EXPERIMENTAL
Preparation of Blood Filtrates-The laked blood filtrates were
prepared according to the well known Folin-Wu procedure.
The unlaked blood filtrates were prepared according to the
directions published by Folin (4) in 1930 with the one exception
that the precipitated proteins were filtered off instead of centri-
fuged. To be able to filter the precipitated proteins is a distinct
advantage when a series of blood filtrates is prepared simultane-
ously. For this filtration it is necessary to use a good grade of
filter paper,’ folded (fluted) so as to expose a large filtering surface.
It is essential that the tip of the folded paper should fit tightly into
the stem of the funnel. Under these conditions filtration is rapid
and a maximum yield of water-clear filtrate (37 to 38 cc. from 5
cc. of blood) is obtained in about 15 minutes. The filtration of
f We have found Schleicher and Schiill No. 597 filter paper very satis-
factory. Whatman No. 41 filter paper permits rapid filtration but we
have found that this paper contains enough ammonia to become a dis-
turbing factor in the amino acid nitrogen determination.
514 Amino Acid Nitrogen in Blood
the unlaked blood should not be continued for more than 20 min-
utes, for on longer standing the red corpuscles begin to disintegrate
due to the excess acid present. The filtering of the precipitated
proteins from laked blood is not as rapid nor is the yield of filtrate
as large, usually 25 to 28 cc. from 5 cc. of blood.
Experiments on Addition of Amino Acids to Whole Blood in
Vitro-The amino a,cids were added to the whole blood by two
methods: (1) by adding to the blood a small volume of an isotonic
salt solution containing the equivalent of 10 mg. per cent of amino
nitrogen, and (2) by adding a weighed amount of solid amino acid
equivalent to 10 mg. per cent. Both methods gave equally good
results.
1 cc. of the amino acid solution containing 10 mg. per cent of
amino nitrogen or the weighed amount of solid amino acid was
introduced into a 250 cc. beaker. Into a second 250 cc. beaker were
measured 100 cc. of blood.2 The blood was then poured rapidly
but gently into the beaker containing the amino acid. After
the blood was gently poured back and fourth between the two
beakers a number of times to insure complete mixing, it was poured
into a 300 cc. Erlenmeyer flask. The flask was stoppered and
placed in a warm water bath (37’) for 1 hour and gently shaken at
intervals during this period.
The amino nitrogen content of the plasma and of the whole blood
in both laked and unlaked blood filtrates was determined by the
modified calorimetric method. From these data the content of
amino nitrogen in the red corpuscles was calculated. Table II
gives a summary of the values found.
It is seen from these values that in all but three cases the re-
covery of the added amino nitrogen was better than 96 per cent,
the poorest was 91.7 per cent. The increase in the amino nitrogen
content of the red corpuscles, calculated from both laked and
unlaked blood filtrate analysis, is comparable in each experiment
except one. In this case the amino nitrogen recovered in the
2 Samples of sheep, dog, and chicken blood were used. The blood
samples from sheep and chicken were obtained fresh from the abattoir and
were generally about 45 minutes to an hour old before they reached the
laboratory and work upon them started. The dog blood was obtained
in large quantities from the femoral artery in conjunction with other ex-
periments.