Use of Ferric Ammonium Sulfate in Serum
Cholesterol Determination
Hsien-wen Feng, Chung-miu Pi, Rong-bor Wang, and Li-chuen Chen
Ferric ammonium
sulfate,
which
over ferric chloride in that it is not
does not evolve hydrogen chloride
sulfuric acid, was used in the ferric
acid
method
Comparable
salts.
for serum
results
are
has advantages
deliquescent and
upon addition of
chloride-sulfuric
cholesterol
obtained
determination.
with
these
iron
al.
(3).
measured
otherwise
Unless
stated,
the
resulting
color
was
at 550 nm.
Results and Discussion
Ferric ammonium
sulfate color reagent was reacted
with cholesterol standard solution (200 ig per 5 ml of gla-
cial acetic acid) to give a plateau of absorbance,
at 540-
560 nm (Figure 1). Absorbance at 550 nm did not change
significantly
within 15-90 mm
afterthe color reagent was
To determine cholesterolin biologicalfluids,use of the
ferric chloride-sulfuric
acid reagent introduced
by Zlatkis
et al. (1) is relatively
simple and reliable (2, 3), and the
sensitivityof the reagent for cholesterol is four to five
times greater than that of the Liebermann-Burchard
reagent (2).
However, Parekh and Jung (4) reported that hydrogen
chloride is evolved during the ferric chloride-sulfuric
acid
reaction, leading to poor control of reaction temperature.
In addition,
the very deliquescent ferric chloride is tedious
added (Table 1). Absorbance was linearly related to cholesterol concentration (Figure 2).
Results of recovery studies were satisfactory: 96-106%,
with a mean of 101.5% (Table 2).
Lemberg
(5) demonstrated that oxidation of bilirubin
gives green-colored biliverdin in the presence of ferric
chloride.
Ferric ammonium
sulfate color reagent behaves
similarlywith bilirubinsolution(100 zg per 5 ml of glacial
acetic acid). The green solution absorbs significantlyat
550 nm (Figure 1). Thus, as in the case of ferricchloride
to handle. The ferricchloride-sulfuricacid method would
thus be better if a nondeliquescent iron salt could be substituted
that forms no volatile
compounds
on reaction
with sulfuric acid. We studied the possibility
of using ferric
ammonium
sulfate.
Materials and Methods
Ferric ammonium
sulfate
stock solution.
Dissolve
4.463
g of ferricammonium
sulfate (FeNH4(S04)2-12H20,
ACS
reagent; Merck and Co., Rahway, N.J.),
in 100 ml of 85%
phosphoric acid (G. R., E. Merck, Darmstadt, Germany).
The solution is stable at room temperature.
Ferric ammonium
sulfate color reagent. Mix 8 ml of the
stock solution with 92 ml of concentrated sulfuric acid
(97.7%, AR grade; J. T. Baker Chemical Co., Phillipsburg,
N.J.
08865). This solution is usable for at least two
months, if protected from atmospheric moisture by a
drying bottle of silica gel.
Bilirubin solution. Dissolve
2 mg of bilirubin(reference
Lu
C-)
z
4
0
C’) 0.
4
0.
WAVELENGTH
Fig.
1. Absorption
nm
curve
standard; Pfanstiehl Labs. Inc., Waukegan, Ill.60085) in
100 ml of glacial acetic acid (mm. 99%, extra pure reagent;
Shimakyu’s
Pure Chemicals, Osaka, Japan).
Ferric
chloride
color
reagent.
Rosenthal et al. (3) with ferric
A.C.S. reagent grade).
Stock cholesterol
ol (reagent grade;
Prepared
according
to
Table 1. Development and Stability of Color
(FeC13#{149}6H20,
Formed by the Reaction of Serum Cholesterol
with Ferric Ammonium Sulfate Color Reagent
100 mg of cholester-
chloride
solution.
Dissolve
Wilson Labs,
Chicago,
Ill.
glacial acetic acid to make a final concentration
60609)
in
of 1.0 g/
liter.
Standard
cholesterol
solution,
100 ig/5 ml. Mix 2 ml of
the stock solution with 98 ml of glacial acetic acid.
Cholesterol
determination.
Cholesterol was extracted
from serum with acetone/alcohol
mixture
(2) and determined with ferric chloride or ferric ammonium sulfate
color
reagent
according
to the procedure
of Rosenthal
et
Mm after adding
color reagent
5
General Hospital, Taipei, Taiwan, Republic of China.
Received Aug. 14, 1972; accepted Oct. 25, 1972.
Cholesterol,
mg/i00 ml
20
35
0300a
0.311
0.310
0.315
249
249
252
40
0.312
250
45
0.314
0.310
0.300
251
247
240
15
90
300
From the Department of Biochemistry,
National
Defense Medical Center; and Kohlberg Medical Research Laboratory, Veterans
A550 nm
#{176}Average
ofthreeseparatedeterminations.
5Cholesterol concentrations
were calculated from the corresponding
absorbances of sample solutionsand the absorbance of the standard
read 30 mm afteradding colorreagent.
CLINICAL
CHEMISTRY,
Vol. 19, No. 1, 1973
121
320
Table 2. Recovery Study on Cholesterol by
Ferric Ammonium Sulfate Method
Eo>
280
Cholesterol
0’
240
E”
Found afteraddition
Presentin
original serum
of cholesterol#{176}
200
E
Recovery, %
oc
60
mg/i 00 ml
283
393
350
340
325
300
302
389
320
259
188
235
240
235
205
193
293
210
158
85
103
106
100
96
98
105
97
105
101
104
Mean
101.5
SE
±1.17
U)
w
-J
o
IC.)
20
80
40
0
7
40
80
20
60
200 240
CHOLESTEROL
(ferric
mg/Ioo
280
320
ml
chloride)
Fig. 3. Correlation between serum
cholesterol as measured by the ferric chloride and ferric
ammonium
sulfate
methods
aloo mg added per 100 ml.
then dissolved in acetic acid to prepare a modified iron
reagent for cholesterol
determination
(4, 8). Our results
suggest that, instead of the very deliquescent
ferric chloride, the nondeliquescent
ferric ammonium
sulfate may
be used to prepare the modified reagent, or even that the
reagent be prepared directly from ferric hydroxide,
which
is also nondeliquescent.
E
0
I’)
I0
Ui
We acknowledge the financial support of the China Medical
Board of New York, and the National Science Council, Republic
C-)
of China.
U)
References
z
4
0
U)
U)
1. Zlatkis, A., Zak, B., and Boyle, A. J., A new method for the
direct determination
of serum cholesterol.
J. Lab. Clin. Med. 41,
0I
4
486(1953).
2. Zak, B., Dickenman, R. C., White, E. G., Burnett,H., and
Cherney, P. J.,Rapid estimationof free and totalcholesterol.
20
250
CHOLESTEROL
Fig. 2. Absorbances
Amer.
mg/looml
of different
cholesterol
concentra-
tions
color reagent
(6, 7), bile pigment
may interfere with cho-
lesterol determination.
Figure 3 indicates that the modified and former procedures give essentially the same results. In general, our results
would
suggest
that
the more
convenient
monium sulfate may be validly substituted
ride in the cholesterol determination.
ferric
am-
for ferric chlo-
Alternatively,
to avoid hydrogen chloride formation,
fernc chloride may be converted to ferric hydroxide,
which is
122 CLINICAL CHEMISTRY, Vol.19,No.1,1973
J. Clin. Pathol.
24, 1307(1954).
M. L., and Buscaglia,S.,A stable
iron reagent for determination of cholesterol. J. Lab. Clin. Med.
50, 318 (1957).
4. Parekh, A. C., and Jung, D. H., Cholesterol determination
with ferricacetate-uraniumacetateand sulfuric
acid-ferrous
sul3. Rosenthal,
fate reagents.
H. L., Pfluke,
Anal.
Chem.
42, 1423(1970).
5. Lemberg, R., Bilirubin
pigments. IV. Dehydrobilirubin.
Ann.
499,25(1932).
6. Zak, B., Weiner,
L. M., and Welsh,
B., Spectrophotometric
study of bilirubin
interference
in the Huang
reaction
for cholesterol. Clin. Chim. Acta3O, 697(1970).
7. Zak, B., and Epstein, E., A study of several colorreactions for
the determination
of cholesterol.Clin. Chim. Acta 6, 72 (1961).
8. Jung, D. H., and Parekh,
A. C., New color reaction
for cholesterol assay. Clin. Chim. Acta 35,73(1971).