colorimetric determination of phospholipids

8/17/2019 Colorimetric Determination of Phospholipids

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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

colorimetric determination of phospholipids

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