ANALYTICAL

BIOCHEMISTRY

Colorirnetric

104,

lo-

14 (1980)

Determination
of Phospholipids
Ferrothiocyanate

with Ammonium

JOHN CHARLES MARSHALI. STEWART

Dcptrrtmrnt

of Child

Hcolth.

iiing'~

Collr~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 conventional acid digestion and color development procedures by forming a complex with ammonium ferrothiocyanate.

Of the many methods that exist for measuring phospholipids

(I-4) those based on
analysis of the phosphorus content presently appear to be most favored. This requires acid digestion of the phospholipid
and calorimetric
determination
of the inorganic 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 phospholipids which eliminates the need for acid
digestion and color development.
Calorimetric methods based on the complex 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) methods, which avoid acid digestion,
have
been proposed. The fluorimetric method is
particularly
sensitive allowing measurement of phospholipid
in the range O.Ol100 pg, but it needs a fluorimeter for use,
and may suffer from quenching effects. The
calorimetric method, while avoiding acid digestion maintains the need for color development. We report here a simpler and
0003.'697i80/07OOIO-05$02.00/O
Copyrtght
F 1980 by Acadomtc Presr. Inc.
All nphf? of reproductmn
in any form reserved.

more rapid method based on complex

formation
between ammonium
ferrothiocyanate 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 distilled 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~.~
were obtained from
Koch-Light
Laboratories,
Colnbrook.
Buckinghamshire.
L-3-Lecithin,
DL-3-lecithin, 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 lysoI0

COLORIMETRIC

DETERMINATION

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 obtained 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 determined by the same laboratory using the
procedure of Saliman ( 14).
Calibration

Graph of Dipalmi~oyl

Lecithin

A solution of 10.0 mg dipalmitoyl

lecithin 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 separating. the lower chloroform phase was removed with a Pasteur pipet (or syringe).
clarified if necessary with a pinch of anhydrous sodium sulfate, and the optical density of the chloroform read at A 488 nm in a
l-cm beam l-cm cuvette and the average
OD plotted (Fig. I).

11

OF PHOSPHOLIPIDS

Analysis of Rat Lil-er Microsomes

Phospholipid

Jbr

(a) By ammonium ferrothiocyanute

camp1e.r formation.
Duplicate 60-. 45-, 30-, and
IS-p1 samples of rat (male Wistar 410 g)
liver microsomal suspension of protein concentration 10.9 mg/ml were extracted with
chloroform and methanol according to Albrinks 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 50C. 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 intimately mixed for 1 min on a rotamixer.
Following
phase separation
the lower
chloroform
phase was removed with a
Pasteur pipet and the optical density measured at A 488 nm in a l-cm beam smallvolume cuvette. The average OD was
plotted (Fig. 2). This gave a value of 413
pmol phospholipid/lOO/~l
of microsomes.
(h) Bx inorganic~ phosphare detertnination. Duplicate 50-~1 aliquots of the microsomal fraction were extracted. digested to
inorganic phosphate, and measured colorimetrically using Albrinks method (12). This
gave a value of 409 pmol of phospholipid
in 100 ~1 of microsomes.
Anulysis ofHemo/yzed
Phospholipid

Blood jitt

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 repeated 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 dissolved in 30 ml chloroform
and mixed

12

JOHN

CHARLES

MARSHALL

intimately with 30 ml ammonium ferrothiocyanate 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 dryness 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

STEWART

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-Lamberts
law is
not obeyed, and there is progressive deviation 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 chloroform 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 involved depending on conditions and solvent.
Our complex, isolated on a preparative run
analysis for a Fe(SCN), species of composition dipalmitoyl
lecithin:Fe(SCN),
1: 1.
The following phospholipids also form a
chloroform
soluble
complex
with ammonium
ferrothiocyanate:
L-3-lecithin,
sphingomyelin,
lysolecithin,
phosphatidyl

AND DISCUSSION

The red inorganic compound ammonium

ferrothiocyanate is insoluble in chloroform,
but forms a complex with dipalmitoyl
lecithin which is freely soluble in chloroform.
When a solution of chloroform containing
dipalmitoyl lecithin is mixed intimately with
ammonium ferrothiocyanate
at room temperature, a colored complex (A,,, 488 nm)
is formed which partitions in the chloroform
phase. A calibration graph was prepared by
mixing ammonium
ferrothiocyanate
solution (2 ml) with chloroform (2 ml) contain-

/
0.4 -

0 /

0.3
0 /
OPTICAL
DENSITY
488 nm

/
o-2 -

OJ/
0

-02

-04
mgr

FIG.

1. Calibration

graph

of dipalmitoyl

DIPALMITOYL

lecithin:

-06

08

,I0

LECITHIN

ammoniumferrothiocyanate

complex.

COLORIMETRIC
TABLE
COMPLEX

DETERMINATION

FORMATION

OF AMMONIUM
WITH

&I,, of
lipid
Fe( SCN jB
complex

Phospholipid

ODimg
(x10-)

ODimol
(X IO 1

Dipalmitoyl

lecithin
L-3-Lecithin
Lysolecithin
Sphingomyelin
Phosphatidyl
serine
Phosphatidyl
ethanolamine

488
485
47.5
490

0.419
0.410
0.530
0.400

0.398
0.395
0.385,
0.3731

452

0.190

0.323

470

0.230

0.371

I In 2.0 ml of chloroform
solution.
h Based on I:1 Fe(SCN),:phospholipid
c Assuming
a I:1 Fe(SCN),,:phospholipid
Assuming
a I:? Fe(SCNI:l:phospholipid

13

PHOSPHOLIPIDS

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 sphingomyelin give a slope which is essentially the
same (Table 1) as that for synthetic dipalmitoyl lecithin, and probably form similar
1: I complexes with FetSCN),. Phosphatidyl
ethanolamine
and phosphatidyl
serine,
however, give a smaller slope, which corresponds more closely to a complex ofcomposition FetSCN),,: phospholipid
1:2.
Currently we are measuring the phospholipid content of biological fluids with ammonium ferrothiocyanate.
Figure 2 shows
the results obtained from an analysis of rat
liver microsomes. Aliquots (15-60 ~1) of a
microsomal
sample were extracted, concentrated to dryness, and the phospholipid
content of the dried extract measured as
described (Experimental).
The standard
reference graph (Fig. 1) was used to determine the phospholipid
content (as dipalmitoyl lecithin) of each aliquot. A value
of 413 pmol phospholipidilO0
~1 of rat liver
microsomes was obtained. The same sample

FERROTHIOCYANA-TE
PHOSPHOLIPIDS

OF

complex.
complex.
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

l /
1

OPTICAL
DENSITY
488nm

02

O-l-

0///

0 li..//,
0

15

30
pmls

FIG.

2. Analysis

A5

MICROSOMES

of rat liver

microsomes.

60

14

JOHN

CHARLES

MARSHALL

analyzed by digestion to inorganic phosphate 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 following advantages.
(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
ferrothiocyanate.
(b) The method is free from interference.
Reproducibility
is excellent on any particular sample. Frequently repeated analyses gave identical optical densities, and the
coefficient variation on analysis of 10 samples of hemolyzed blood was 6.05%. Inorganic phosphate and free fatty acids do
not interfere with the formation
of ammonium ferrothiocyanate complex and solution blanks are zero.
(c) Ammonium
ferrothiocyanate
method
is useful for measuring phospholipids which
are present singly or as mixtures in solution. In the latter cases it will not characterize individual phospholipids.

STEWART

ACKNOWLEDGMENTS
I thank Dr. H. R. Gamsu for his advice and encouragement
throughout
this work.
and Dr. D. .I. Fry
(Anatomy
Dundee)
for providing
the rat liver microsomal samples.
Dr. Margaret
Brothwood
analyzed
the hemolyzed
blood samples.
This work
was supported by a grant from the Wates Foundation.

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of Phospholipids
(Ansell, G. B.. Hawthorne,
J. N.. and
McDawson.
R.. eds.),
Vol. 3, Chap. 3, pp.
43-65.
BBA Library
Elsevier,
Amsterdam.
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R. L. (1969) Diagnostic
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