A Rapid Spectrophotometric
Method for the
Determination
of Citric Acid in Blood
Rennie J. Camp and Lyneffe
Farmer
A method is described for a determination of citric acid in serum. This method is
based on the formation of pentabromoacetone from citric acid and subsequent color
development with thiourea.
IN
AN INVESTIGATION
metabolism
of citric
of the possible
acid,
lactic
effects
acid,
and
of transaconitic
pyruvic
acid
acid
on the
in mammalian
species, it became necessary
to develop
a rapid and reliable
procedure
for the determination
of citric acid in blood.
Several
methods
have been developed
for tile determination
of citric
acid in biologic fluids. Iii all of these procedures,
citric acid is converted
to pentabromoacetone,
and the pentabromoacetone
is measured
spectrophotometrically
using one of the following:
sodium sulfide (1), sodium
iodide
(2), thiourea
(3), potassium
iodide
(4),
or pyridine
(5). The
reliable
and sensitive
procedures
are tedious
and laborious,
and the
more rapid methods
lack specificity
arid reproducibility.
The procedure
for citric acid described
in this paper
is an attempt
to incorporate
the reliable and sensitive
technics
of the time-consuming
methods
with tile more rapid technics
of the less reliable
procedures
to
give a rapid and reliable
procedure
for citric acid.
Materials and Methods
Reagentsand Apparatus
All solutions
were prepared
in triple-distilled
1. Glassware
Glass-stoppered
test tubes
9810).
Glassware
was washed
in clIromic
acid
triple-distilled
water.
2. Vortex mixer
3. Standard
citric acid solution
Dissolve
or deionized
water.
(13 ml., Corning
No.
solution
and rinsed
in
200 mg.
of anhydrous
From
the Department
of Biochemistry
and Biophysics,
Texas
A&M University,
Station,
Tex. 77843.
Aided in part by a grant from the Herman
Fraseh
Foundation,
New York, N. Y.
Received for publication Dec. 7, 1966; accepted
for publication
Feb. 6, 1967.
501
College
502
citric
CAMP & FARMER
acid
Clinical
in 20 ml. of 9 N 112504 (1 :4 dilution
of colIc. reagent
to 200 ml. with water.
Solution
contains
1000 g./lnl.
Chemistry
acid)
and dilute
Store
in refrigerator.
4. Working
citric acid solutions
A. 200 tg./ml.
Pipet 20 ml. of standard
citric acid solUtiOIl into
a 100-ml. volumetric
flask and dilute to volume. Store in refrigerator.
B. 100 g./ml.
Pipet 10 ml. of standard
citric acid solution
into
a 100-mi. volumetric
flask and dilute to volume.
Store in refrigerator.
5. Potassium
permanganate-sodium
bromide
solution
Dissolve
5.0 gm. KMnO4 in 85.0 ml. of boiled, cooled water. Add 5.0 gm. NaBr
with stirring.
Dilute to 100-rnl. volume.
Solution
is stable for several
weeks if stored in the dark in a glass-stoppered,
brown bottle.
6. Trichioroacetic
acid, 15% (w/v)
7. Sulfuric
acid-metapltospboric
acid
I)issolve
5.0 gm. HPO3 in
50 ml. of 9 N 112S04. Stable.
8. Hydrogen
peroxide,
3%
9. Thiourea
solution
(Eastman)
pH 9.2
Dissolve
2.0 gm. of
sodium borate in 100 ml. of 4% (w/v)
thiourea
solution.
Stable.
10. n-He ptane
Baker reagent
grade.
Procedures
Deproteinizafion
To 1.0 ml. of serum add 1.0 ml. of 15% TCA. Agitate
on a vortex
mixer, and allow to stand for 5 mm. Centrifuge
for 15 mm. at 2000 rpm,
and decant
the supernatant
phase.
A second
centrifugation
may be
necessary
with some samples.
Peniabromoacefone
Formation
To 1.0 ml. of the
stoppered
test tule
protein-free
is added
sample
or working
standard
in a glass-
1.0 ml. of the sulfuric
acid-metaphosphoric
acid solution.
riheil the test tube is placed in an ice bath. While gently
shakimig the tube, 2.0 ml. of the KMnO4-NaBr
solution
is added, and the
tube is returned
to the ice bath for 10 miii. After 10 mm., 3% 11202 is
added
dropwise
best accomplished
until
tile
solution
becomes
colorless.
This
by adding
the 11900 dropwise
until
a
color is attained.
If the solution
does not become colorless
additional
H000 is added
dropwise
until
the desired
reached.
Added to the tube is 1.5 ml. of n-heptane;
the
agitated
on tile vortex
mixer for 30 sec. to extract
the
acetone from tile aqueous
phase.
end-point
is
faint,
yellow
in 2-3 mm.,
end-point
is
tube is then
pentabromo-
Color Development
Transfer
a 1.0-mi. aliquot of the n-heptane
layer
test tube, and add 3.5 ml. of the thiourea
solution.
to a glass-stoppered
Agitate
the tube
on
CITRIC ACID IN BLOOD
Vol. 13, No. 6, 1967
503
the vortex
mixer at highest
speed for 1 mm. Transfer
the aqueous
phase to a cuvet and read oii a Coleman
spectrophotometer,
Model 61),
at 445 mp. The blank is prepared
by shaking
1.0 ml. of n-heptane
with
3.5 ml. of thiourea
solution.
The intensity
of the color was read on the
per cent transmission
scale and absorbance
was taken from a table.
Results and Discussion
A typical calibration
curve is presented
in Fig. 1. A linear relationship between
absorbance
alid concentration
is foumid up to 70 pg. citric
acid/ml.
Recovery
studies
of citric acid added
to serum
were conducted
to
measure
the precision
of the mnethod at different
levels of concentration.
\arious
amounts
of citric acid were added to 1.0-ml. samples
of serum.
Then the volume was adjusted
to 5.0 ml. with TCA and a l.0-ml. aliquot
of the protein-free
solution
was treated
in the manner
described
under
Fig.
curve
oh
1.
for
Coleman
Model
Typical
citric
calibration
acid
obtained
spectrophotometer,
6D.
Materials
and Methods. The results
are summarized
in Table 1. The
desirable
sensitivity
range for most galvanometers
is between
80 and
15% transmission.
In samples
where the color intensity
is above 80%
tralIsmission
using this procedure,
it \\‘OUl(i i)e desirable
to make certain
504
CAMP & FARMER
Table
1. RECOVERY
OF KNOWN
AMOUNTS
OF CITRIC
Clinical
ACID
ADDED
TO SERUM
DupUcate
Original
18.5
21.5
18.5
21.5
Added
18.5
21.5
18.5
6.25
6.25
12.50
12.50
25.00
25.00
37.50
21.5
37.50
Calculated
24.75
27.75
31.00
34.00
4350
46.50
56.00
59.00
Found
Chemistry
(pg./ml.)
determinations
Recovery
(%)
26.0,
26.5,
31.0,
30.5,
43.5,
45.5,
57.0,
26.5
27.5
31.0
35.0
43.5
48.5
57.0
105,
96,
100,
90,
100,
98,
102,
107
99
100
103
100
104
102
60.0,
60.0
102, 102
modifications
in the procedure
to obtain a color intensity
which would
fall within the desirable
sensitivity
range. The modifications
which are
available
for this procedure
include:
(1) increasing
the protein-free
filtrate
2 to 6-fold;
(2) using a smaller
volume of thiourea
solution
for
developing
the color;
(3) using a cuvet with a longer light path such
as the 40-mm. microcuvet
for the Klett Summerson
colorimeter;
or (4)
using a combination
of tile aforementioned
modifications.
No measurable
effects were found on the recovery
of added citric acid
(100 pg./ml.)
to serum when the following
possibly
interfering
compounds
were also added
to the serum
sample:
acetone,
20 pg./ml.;
transaconitic
acid, 20 pg./ml.;
glucose,
100 g./mI.;
lactic acid, 40 pg./
ml.; $-hydroxybutyric
acid, 20 /Lg./ml.; and pyruvic
acid, 20 pg./ml.
Lane and Chen (6) developed
a rapid colorimetric
determination
of
citric acid which utilizes
the formation
of iodine
from pentabromoacetone and sodium iodide in an acid solution.
Tn this method, citric acid
is converted
to pentabromoacetone
Ill
the presence
of potassium
permanganate
and potassium
bromide,
and the excess permanganate
is
decolorized
with hydrogen
peroxide.
Tf an excess of hydrogen
peroxide
is added, it will liberate
iodine from sodium
iodide when the color is
developed
with pentabromoacetone
and thus give erroneous
results.
The procedure
of Natelsori
et al. (3) for the formation
of pentabromoacetone
is time consuming;
however,
their colorimetric
determination
of pentabromoacetone
using thiourea
as tile color reagent
is sensitive
and reliable.
In their procedure,
the blank and unknown
solutions
must
be read at different
wave lengths
to correct
for cloudiness
between
these 2 solutions.
Conclusions
The procedure
described
in this l)apeI has incorporated
the rapid
technics
of Lane and Chen (6) for tile formation
of pentabromoacetone,
arid the colorimetric
determination
of pentabromoacetoTle
by Natelsoii
Vol. 13, No. 6, 1967
CITRIC
ACID
IN BLOOD
ci al. (3). The color development
is rapid and
2 hr. As many as 15-20 assays can be con(iucted
The procedure
caii be adapted
to microprocedures
sensitivity
or reliability
of the method.
505
stable for a period
of
within a 2-hr. period.
without
altering
the
References
1.
Natelson,
S., Lugovoy,
J. K., antI Puicus,
J. B., 1)eterminatioii
of micro quantities
of citric
acid in biological
fluids. J. Biol. Chem. 170, 597 (1948).
2. Taussky,
H. H., and Shorr, E., A mierocolorimetric
method
for the determination
of citric
acid. J. Biol. Che,n. 169, 103 (1947).
3. Natelson, S., Pincus, J. B., and Lugovoy, J. K., Microestiination of citric acid; a new
colorimetric reaction for pentabromoacetone. J. Biol. Cheat. 175, 745 (1948).
4. Wolcott, G. H., and Boyer, P. D., A colorimetric method for tiledetermination
of citric
acid in blood and plasma.
J. Bid. Cheat. 172, 729 (1948).
5. Ettinger,
R. H., Goldbaum,
L. R., and Smith,
L. H., A simplified
photometric
method
for
the determination
of citric
acid in biological
fluids.
J. Biol. Chem. 199, 531 (1952).
6. baste, K., and Chen, P. 5., Jr., Rapid
determination
of citric
acid in biological
fluids.
Atomic
Energy
Project
UR579,
The Iniversity
of Rochester,
Aug. 29, 1960.