QUANTITATIVE DETERMINATION

OF PHOSPHOLIPIDS

The estimation of the total amount of phospholipids in a tissue extract must preferably be estimated before any chromatographic separation. This prevents the inevitable loss of phospholipid molecules on adsorbents and thus allows accurate estimation of the ratio of cholesterol to phospholipids in cellular extracts.
We propose below a simple method to estimate rapidly phospholipids in lipid extract and two methods based on the estimation of the amount of phosphorus in extracts or chromatographic fractions. A nondestructive method base on NMR spectroscopy was developed for the determination of phospholipids (Hatzakis E et al., J Agric Food Chem 2008, 56, 6232). This technique, involving a specialized instrumentation, enables also the determination of the fatty acid pattern of these phospholipids.


1. Direct estimation of phospholipids

This colorimetric method is based on the formation of a complex between phospholipids and ammonium ferrothiocyanate (Stewart JCM, Anal Biochem 1980, 104, 10).

Reagents:

Dissolve 27 g of ferric chloride (FeCl₃ 6H₂O) and 30 g of ammonium thiocyanate (NH₄SCN) in 1 liter water. This solution is stable for months at room temperature.
Standard solution of phosphatidylcholine in chloroform (1 mg/ml).

Procedure:

Evaporate completely aliquots of phospholipid solutions in glass tubes.
Dissolve the phospholipid residue in 2 ml chloroform and add 1 ml of thiocyanate reagent. Vortex 1 min and centrifuge at low speed, remove the red lower layer (chloroform) with a Pasteur pipette.
Read the absorbance of this solution at 488 nm and compare with known amounts of a standard phospholipid solution (range: 10-100 µg per tube).
All phospholipids do not give the same response but this method is useful for measuring rapidly phospholipids in mixtures before further analyses.

2. Phospholipid determination by phosphorus assay

Phospholipids in lipid extracts or in chromatographic fractions are satisfactorily estimated by phosphorus determination through an acidic digestion. The released inorganic phosphate is reacted with ammonium molybdate, the complex giving a strong blue color.
Two methods are proposed below according to the sensibility required.

A. Phosphorus assay for amounts higher than 1-2 µg (25-50 µg phospholipids)
(Rouser et al., Lipids 1970, 5, 494-6)

Apparatus:

Electrically heating block set at 180°C. Boiling water bath.
Glass tubes (to be centrifuged) washed before use in water acidified with conc. nitric acid (about 1% final) and dried in an oven at 100°C.

Reagents:

conc. perchloric acid (70%)
Ammonium molybdate solution (2.5 g in 100 ml water)
Ascorbic acid solution (10 g in 100 ml water). Prepare freshly (don't keep more than one week at 4°C).
Standard solution of KH₂PO₄: dilute 10 times a stock solution of KH₂PO₄ (439 mg per liter water, i.e. 100 µg P/ml).

Procedure:

Lipid samples are transferred into clean glass tubes and the solvent is completely evaporated. Add 0.65 ml perchloric acid and place the tubes in the heated block for about 30 min or until the yellow color has disappeared. Silica gel spots may be digested in the same way.
When cool, add to the tubes 3.3 ml water, 0.5 ml of molybdate solution and then 0.5 ml of ascorbic acid solution. Agitate on a vortex after each addition.
The tubes are placed in a boiling water bath for 5 min.
The absorbance of cool samples (including the standards) are read at 800 nm
Standards (1 to 5 µg P/tube) are diluted in 3.3 ml water and 0.65 ml perchloric acid. Digestion is not necessary before adding reagents.
Classically, 5 µg P give an absorbance of 0.9
The amount of phospholipids is calculated directly on a molar basis from the amount of P and on a weight basis after multiplying the amount of P by 25.

In the presence of silica gel (scraped from TLC plates), centrifuge the tubes at low speed and remove the colored solution before measuring its absorbance.

B. Phosphorus assay for amounts lower than 1 µg (25 µg phospholipids)

An improved method was developed for the determination of lipid phosphorus in the sub µg range (Zhou et al., J Lipid Res 1992, 33, 1233). This method is based on the formation of a complex between phosphomolybdenum and malachite green and its resultant shift in absorption maximum. It is convenient for the determination of phospholipid distribution after TLC.

Apparatus:

Electrically heating block set at 180°C.
Glass tubes (to be centrifuged) washed before use in water acidified with conc. nitric acid (about 1% final) and dried in an oven at 100°C.

Reagents:

conc. perchloric acid (70%)
Solution A: 0.4% malachite green (w/v) in water. Prepare by vigorously stirring with a magnetic stirrer for 30 min.
Solution B: 4.2% ammonium molybdate (w/v) in 5M HCl. Prepare by vigorously stirring with a magnetic stirrer for 30 min.
Solution C: 1.5% Tween 20 (w/v) in water. Prepare by vigorously stirring with a magnetic stirrer for 30 min.
Solution D: mix 1 vol. solution B with 3 vol. solution A. Prepare by vigorously stirring with a magnetic stirrer for 30 min. Filter through Whatman paper to obtain a yellow clear solution. This stock solution is stable 2-3 weeks in the dark.
Working solution: before use, mix 1 ml of solution C with 32 ml of solution D. Prepare by vigorously stirring with a magnetic stirrer for 30 min.
Standard solution of KH₂PO₄: dilute 20 times a stock solution of KH₂PO₄ (439 mg per liter water, i.e. 100 µg P/ml).

Procedure:

A. Lipid samples without silica gel

Lipid samples are transferred into clean glass tubes and the solvent is completely evaporated. Add 0.2 ml perchloric acid and place the tubes in the heated block for 30-60 min or until the yellow color has disappeared.
When cool, add to the tubes 0.2 ml water and 2 ml of working solution. Agitate on a vortex and measure the absorbance after 20 min at 660 nm..
Standards (0 to 0.2ml) are diluted to 0.2 ml before adding 0.2 ml perchloric acid.
Classically, 0.6 µg P give an absorbance of 1.

B. Lipid samples with silica gel (scraped spots)

Scraped spots are transferred into clean glass tubes, add .2 ml water and 0.4 ml perchloric acid. Heat at 180°C. When cool, centrifuge at low speed and collect 0.2 ml of the supernatant for phosphorus determination as in A. Multiply the measured value by 2 to get the total amount present in the tube.

Comments :

A modified version of the malachite procedure was reported to be relevant for the determination of total phosphorus in vegetal oils (Szydlowska-Czerniak A et al., Food Chem 2003, 81, 613). After mineralization of oil samples (up to 26 g) with MgO at 800°C, the malachite green method allowed the estimation of the very low levels of lipidic phosphorus found in refined oils (about 2 mg per Kg). 

3 - Estimation of phospholipids from fatty acid amounts

A quantitative determination of the amount of fatty acids in a phospholipid sample enables a more precise estimation of the amount of phospholipid than with the phosphorus amount. 
To know that amount, the fatty acid weight must be multiplied by 1.3. 
When a definite phospholipid class is analyzed, the value of the coefficient must be adapted to that class.  The table below gives these values for the main phospholipid classes :

DETERMINATION OF THE AMOUNT

OF PLASMALOGENS

Three approaches are classically used to quantify plasmalogens (aldehydogenic lipids), namely the estimation of long-chains aldehydes released through an acidic treatment, the determination of the vinyl ether content, and the HPLC separation of intact plasmalogens.

1. Long-chain aldehydes in free or bound forms may be estimated colorimetrically by conversion to nitrophenylhydrazones or by GLC of the dimethyl acetal derivatives.

2. The vinyl ether content of a phospholipid sample can be determined by the iodine uptake method as it was described in its last modification by Gottfried et al (J Biol Chem 1962, 237, 329). The specificity of the determination is based on the more rapid interaction of iodine with vinyl ethers than with olefinic double bonds.

Reagents:

Solution of potassium iodide (3% in water, w/v)
Solution of iodine (7 mg iodine in 10 ml KI solution)
Working solution: mix 1 ml of iodine solution with 9 ml of KI solution

Procedure:

Evaporate an aliquot of lipid solution (50-100 nmol) in a glass tube and dissolve in 0.5 ml methanol. Add 0.5 ml of working solution and mix for 30 min at room temperature.
Add 4 ml of 95% ethanol and read the absorbance at 355 nm against a blank in which 0.5 ml of 3% KI is added to the sample instead of the iodine solution. Run also a control tube in which the lipid sample is omitted. The molar extinction coefficient is about 27 500.

Calculation: µmoles vinyl groups = [(absorbance of iodine control - absorbance of sample) / 27 500] x 5000

Other procedure :

A highly sensitive determination of plasmalogens based on HPLC separation of molecules labeled with radioactive iodine (¹²⁵I) was described (Maeba R et al., Anal Biochem 2004, 331, 169). The method sensitivity is more than 1000-fold higher than the classical determination with nonradioactive iodine.