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ANALYTICAL BIOCHEMISTRY 104, lo- 14 (1980)
Colorirnetric Determination of Phospholipids with Ammonium
Ferrothiocyanate
JOHN CHARLES MARSHALI.
STEWART
Dcptrrtm rnt of‘ Child Hcolth. iiing'~ C‘ollr~c Hospittrl Mrtiicwl S~~hool. DrrvmrX Hill. Londm~ S.E.5.. Gr,g /trnd
Received October IS. 1979
Phospholipids may be measured calorimetrically (as dipalmitoyl lecithin) without conven-
tional acid digestion and color development procedures by forming a complex with am-
monium ferrothiocyanate.
Of the many methods that exist for meas-
uring phospholipids (I-4) those based on
analysis of the phosphorus content pres-
ently appear to be most favored. This re-
quires acid digestion of the phospholipid
and calorimetric determination of the in-
organic phosphate (5,6) formed. This is a
sensitive method allowing the determination
of low levels of phospholipid, but is lengthy.
We were prompted to look for an alternative
more rapid method, and report here a
calorimetric method for measuring phos-
pholipids which eliminates the need for acid
digestion and color development.
Calorimetric methods based on the com-
plex formation (usually a simple salt) of an
ionic substance with a dye of the opposite
charge are extensively used in surfactant
measurements (7,8), but phospholipids have
not been traditionally measured by such
methods although they readily fix the ions
of ordinary salts and dyes (9). Recently
fluorimetric (10) and calorimetric (1 I) meth-
ods, which avoid acid digestion, have
been proposed. The fluorimetric method is
particularly sensitive allowing measure-
ment of phospholipid in the range O.Ol-
100 pg, but it needs a fluorimeter for use,
and may suffer from quenching effects. The
calorimetric method, while avoiding acid di-
gestion maintains the need for color de-
velopment. We report here a simpler and
more rapid method based on complex
formation between ammonium ferrothio-
cyanate and phospholipids which allows
measurements of phospholipids in the range
0.01-o. 1 mg ( 15- 150 nmol).
EXPERIMENTAL
All glassware was cleaned with chromic
acid and well washed with deionized dis-
tilled water before use.
Sodium sulfate, ammonium thiocyanate,
ferric chloride, and chloroform were all of
analytical grade and purchased from British
Drug Houses. Poole, Dorset, England.
All ~~z~~.~phu/~pj~.~
ere obtained from
Koch-Light Laboratories, Colnbrook.
Buckinghamshire. L-3-Lecithin, DL-3-leci-
thin, and t.-3-phosphatidyl ethanolamine
were synthesized materials and were used
as supplied without further purification.
L-3-Lecithin was 95 analytically pure.
or-3-lecithin was 98 analytically pure, and
L-3-phosphatidyl ethanolamine gave a single
spot on thin-layer plate chromatography (R,
0.6) in chloroform:methanol:water (95:35:
5. v/v).
Phosphatidyl-L-serine and sphingomyelin
were obtained from bovine brain, and lyso-
0003.'697i80/07OOIO-05 02.00/O
Copyrtght F 1980 by Acado mtc Presr. In c.
All nphf? of reproductmn in any form reserved.
I 0
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COLORIMETRIC DETERMINATION OF PHOSPH OLIPIDS
11
lecithin was from egg lecithin. Lysolecithin
was prepared by enzymatic hydrolysis of
purified egg lecithin followed by silicic acid
chromatography and gave a single spot on
thin-layer plate chromatography.
Spectrtr
Ultraviolet and visible spectra were ob-
tained on a Unicam SPl800 and Beckman
DB-G spectrophotometer.
Ammonium FclrrothiocvLtnatr
Throughout the work a standard solution
(N/ 10) of ammonium ferrothiocyanate was
used. It was prepared by dissolving 27.03 g
ferric chloride hexahydrate (FeC1,6H20)
and 30.4 g ammonium thiocyanate (NH,SCN)
in deionized distilled water and making up to
1 liter. It is stable for months at room
temperature.
Elrtnet~tal Analysis
Elemental analysis was performed by the
Microanalytical Laboratory. University
College, London, using a Perkin-Elmer
240 elemental analyzer. Phosphorus was de-
termined by the same laboratory using the
procedure of Saliman ( 14).
Calibration Graph of Dipalmi~oyl Lecithin
A solution of 10.0 mg dipalmitoyl leci-
thin in 100 ml chloroform was first prepared.
Duplicate volumes of this between 0.1 and
1.0 ml were then pipetted off, added to 2.0
ml ammonium ferrothiocyanate solution in a
test tube, and enough chloroform was added
to make the final chloroform volume 2.0 ml.
The biphasic system was then vigorously
mixed on a rotamixer for 1 min. On separat-
ing. the lower chloroform phase was re-
moved with a Pasteur pipet (or syringe).
clarified if necessary with a pinch of anhy-
drous sodium sulfate, and the optical den-
sity of the chloroform read at A 488 nm in a
l-cm beam l-cm” cuvette and the average
OD plotted (Fig. I).
Analysis of Rat Lil-er Microsomes Jbr
Phospholipid
(a) By ammonium ferrothiocyanute cam-
p1e.r formation. Duplicate 60-. 45-, 30-, and
IS-p1 samples of rat (male Wistar 410 g)
liver microsomal suspension of protein con-
centration 10.9 mg/ml were extracted with
chloroform and methanol according to Al-
brinks procedure (12). On separating, 1.0
ml of each chloroform extract (total volume
2.6 ml) was removed with a syringe and
concentrated to wtnpletr dryness in a
stream of air at 50°C. The dried extract of
phospholipids was then dissolved in 2.0 ml
chloroform, added to 2.0 ml of ammonium
ferrothiocyanate in a test tube, and in-
timately mixed for 1 min on a rotamixer.
Following phase separation the lower
chloroform phase was removed with a
Pasteur pipet and the optical density meas-
ured at A 488 nm in a l-cm beam small-
volume cuvette. The average OD was
plotted (Fig. 2). This gave a value of 413
pmol phospholipid/lOO/~l of microsomes.
(h) Bx inorganic~ phosphare detertnina-
tion. Duplicate 50-~1 aliquots of the micro-
somal fraction were extracted. digested to
inorganic phosphate, and measured color-
imetrically using Albrinks method (12). This
gave a value of 409 pmol of phospholipid
in 100 ~1 of microsomes.
Anulysis of’Hemo/yzed Blood jitt
Phospholipid
One-milliliter samples of hemolyzed
blood (0.4 mg lithium heparinized blood in
250 ml of water) were extracted and
analyzed for phospholipid. This was re-
peated 10 times and gave a mean value of
0.064 mg in 10 ml 2 0.004 mg in 10 ml.
Coefficient of variation 6.05 .
Dipalmitoyl Lecithin: Ammonium
Ferrothiocyanufe Cotnples
Dipalmitoyl lecithin, 103.6 mg, was dis-
solved in 30 ml chloroform and mixed
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12 JOHN CHARLES MARSHALL STEW ART
intimately with 30 ml ammonium ferrothio-
cyanate solution. The lower chloroform
solution was removed on separating and the
aqueous phase reextracted twice with 30 ml
chloroform. The chloroform layers were
combined, dried with anhydrous sodium
sulfate, filtered, and concentrated to dry-
ness to give 120.7 mgof product (yield 89 ).
Anal.
Calcd for C,,H,,N,P,O,S,Fe: C.
53.6; H, 8.3; N, 5.81, P, 3.22; S, 9.9. Found:
C, 53.73; H. 8.5; N, 6.02; P, 3.98; S, 9.36.
RESULTS AND DISCUSSION
The red inorganic compound ammonium
ferrothiocyanate is insoluble in chloroform,
but forms a complex with dipalmitoyl leci-
thin which is freely soluble in chloroform.
When a solution of chloroform containing
dipalmitoyl lecithin is mixed intimately with
ammonium ferrothiocyanate at room tem-
perature, a colored complex (A,,, 488 nm)
is formed which partitions in the chloroform
phase. A calibration graph was prepared by
mixing ammonium ferrothiocyanate solu-
tion (2 ml) with chloroform (2 ml) contain-
ing increasing concentrations of dipalmitoyl
lecithin and measuring the optical density of
the chloroform phase at h 488 nm. Figure
1 is the plot obtained for concentrations of
dipalmitoyl lecithin up to 0.1 mg in 2 ml of
chloroform, and is linear up to 0.4 OD
unit. Beyond this Beer-Lambert’s law is
not obeyed, and there is progressive devia-
tion of the plot from linearity. Less than 5
pg of dipalmitoyl lecithin may be measured,
and this sensitivity could be improved if
desired by using a smaller volume of chloro-
form in the extraction phase.
The extraction of ferrothiocyanate from
aqueous solution has been studied in some
detail by Maddock (13) who concluded on
spectroscopic grounds that either of the
species FE(SCN), or FE(SCN), may be in-
volved depending on conditions and solvent.
Our complex, isolated on a preparative run
analysis for a Fe(SCN), species of composi-
tion dipalmitoyl lecithin:Fe(SCN), 1: 1.
The following phospholipids also form a
chloroform
soluble
complex with am-
monium ferrothiocyanate: L-3-lecithin,
sphingomyelin, lysolecithin, phosphatidyl
/
0.4 -
0 /
0.3
0 /
OPTI CAL
/
DENS ITY o-2 -
488 nm
/
OJ/
0 -02 -04 -06 08 ,I0
mgr D I PALMI TOYL LECITHI N
FIG. 1. Calibration graph of dipalm itoyl lecit hin: ammo niumferrothiocyanate complex.
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COLORIMETRIC DETERMINATION OF PHOSPHO LIPIDS
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TABLE 1
COMPLEX FORMAT ION OF AMMONIUM
FERROTHIOCYANA-TE WITH
PHOSPHOLIPIDS
Phospholipid
&I,, of
lipid
Fe( SCN jB
ODimg” ODimol”
complex (x10-‘) (X IO ‘1
Dipalmitoyl
lecithin
L-3-Lecithin
Lysolecithin
Sphingomyelin
Phosphatidyl
serine
Phosphatidyl
ethanolamine
488 0.419 0.398”
485 0.410 0.395’
47.5 0.530 0.385,
490 0.400 0.3731
452 0.190 0.323”
470 0.230 0.371”
‘I In 2.0 ml of chloroform solution.
h Base d on I:1 Fe(SCN),:phosph olipid complex.
c Assum ing a I:1 Fe(SCN),,:phospholipid complex.
” Assum ing a I:? Fe(SCNI:l:phospholipid complex.
ethanolamine, and phosphatidyl serine. The
complexes between these phospholipids
and ammonium ferrothiocyanate were not
characterized as for dipalmitoyl lecithin
but calibration graphs were prepared for
all the phospholipids in a similar manner
0.4
1
I
OPTI CAL
DENSITY 02
488nm
i
to that for dipalmitoyl lecithin, and linear
plots were obtained for all between 0 and
0.40 OD unit. Table 1 gives the slopes for
all
the phospholipids studied in OD per
milligram of phospholipid and OD per mole
of phospholipid. Lysolecithin and sphingo-
myelin give a slope which is essentially the
same (Table 1) as that for synthetic di-
palmitoyl lecithin, and probably form similar
1: I complexes with FetSCN),. Phosphatidyl
ethanolamine and phosphatidyl serine,
however, give a smaller slope, which cor-
responds more closely to a complex ofcom-
position FetSCN),,: phospholipid 1:2.
Currently we are measuring the phospho-
lipid content of biological fluids with am-
monium ferrothiocyanate. Figure 2 shows
the results obtained from an analysis of rat
liver microsomes. Aliquots (15-60 ~1) of a
microsomal sample were extracted, con-
centrated to dryness, and the phospholipid
content of the dried extract measured as
described (Experimental). The standard
reference graph (Fig. 1) was used to de-
termine the phospholipid content (as di-
palmitoyl lecithin) of each aliquot. A value
of 413 pmol phospholipidilO0 ~1 of rat liver
microsomes was obtained. The same sample
l
/
/’
O-l ///
0
0’
li..//,
0 15
30 A5 60
pmls MICROSOMES
FIG. 2. Ana lysis of rat liver micros omes .
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14
JOHN CHARLES MARSHALL STEW ART
analyzed by digestion to inorganic phos-
phate give a phospholipid content of 409
pmol/lOO ~1. No special difficulties attend
the practical procedure, however, it is best
to wash glassware with chromic acid to
avoid possible contamination from surface
active cleansing agents; it is also important
to take the chloroform/methanol extract to
complete dryness since traces of methanol
would interfere with the final partition step.
The ammonium ferrothiocyanate method
for measuring phospholipids offers the fol-
lowing advantages.
I thank Dr. H. R. Gamsu for his advice and en-
couragement throughout this work. and Dr. D. .I. Fry
(Anatomy Dundee) for providing the rat liver micro-
som al sam ples. Dr. Margaret Brothwood analyzed
the hemolyzed blood sample s. Th is work was sup-
ported by a grant from the Wate s Foundation.
(a) It is more rapid than methods based
on digestion to inorganic phosphate. A dried
extract of phospholipid may be measured
in less than 10 min using ammonium fer-
rothiocyanate.
I. Spanner, S. ( 1974) in Form and Function of Phos-
pho lipids (Anse ll, G. B.. Hawthorne, J. N.. and
McDawson. R.. eds.), Vol. 3, Chap. 3 , pp.
43-65. BBA Library Elsevier, Amsterdam .
2. Searcy, R. L. (1969) Diagno stic Bioche mistry,
pp. 400-413 . McG raw-Hill, New York.
3. Karlander. S. -G.. Karlss on, K. -A.. and Pasch er.
1. (1973)Eio~.lri~r. f3ioph.v.v. A