Based on the request of many scientists, we are going to post several liposome related protocols on the blog for the next couple of months.
The concentration of the lipids in the liposomes can be measured by spectrophotometric methods. There are two methods that are used in many research labs.
1) The Bartlett assay
2) The Stewart assay
In this post we will explain the Bartlett assay.
The principle of the Bartlett assay is based on the colorimetric determination of inorganic phosphate. The phospholipid content of liposomes can be determined after destruction of the phospholipid with perchloric acid to inorganic phosphate. The inorganic phosphate is converted to phospho-molybdic acid by the addition of ammonium molybdate, which is reduced to a blue colored complex by 4-amino-2-naphthyl-4-sulfonic acid during heating. This compound can be determined colorimetrically at 830 nm.
A Barlett assay modified by Barenholz is presented here.
Reference: Barenholz, Y. and Amselem, S. (1993). Liposome Technology, 2nd edition, Vol. I, p. 527, CRC Press, Boca Raton, FL.
Equipment and reagents
1) 10 -15 ml glass test tubes
2) Boiling stones
3) Marbles to cover the test tubes
4) 180 degree C heating block or oven
5) Cuvettes for spectrophotometer
7) 0.65 mM phosphorus standard solution (Sigma catalog number P3869)
8) 70% perchloric acid (Sigma catalog number 380083)
9) 4-Amino-2-naphthyl-4-sulfonic acid reagent. 0.8 gram of Fisk and Subbarow reducer (Sigma catalog number F5428) is dissolved in 5 ml of double distilled water. Cover the solution container with aluminum foil. The solution will be stable at refrigerator at 4 C and in dark for few months. Precipitation in the solution might occur over time but may not be a problem when using the supernatant.
10) 5% (w/w) ammonium molybdate solution. Dissolve 2.5 gram ammonium molybdate (Sigma catalog number 277908) in 50 ml of double-distilled water.
1) Add 0, 25, 50, 100, 125 microliters phosphorus standard solution and an appropriate amount of the liposome samples in triplicate (containing about 30 nmol phospholipids) in separate 10-15 ml glass tubes.
2) Add two boiling stones in each tube.
3) Add 0.4 ml of 70% perchloric acid.
4) Cover the tubes with marbles and incubate them for 30 min at 180 degree centigrade in a a heating block or pre-heated oven.
5) Cool the tubes to ambient temperature.
6) Add to each tube
a) 1.2 ml water
b) 0.2 ml of 5% (w/w) ammonium molybdate solution
c) 50 microliters amino-naphthyl-sulfonic acid reagent.
7) Vortex each tube and place the tubes in boiling water for 7 minutes.
8) Cool the tubes to ambient temperature and measure the absorbance of the standards and samples at 830 nm.
9) Plot the absorbance against the phosphate concentration to check the linearity of the standard curve and to calculate the concentration of the phospholipids in the liposome sample from the absorbance by reference to the standard curve.
1) The presence of inorganic phosphate in buffers will interfere with phospholipid quantification. Therefore Bartlett assay can not be used when the liposomes are made in PBS or other phosphate based buffers. However Stewart assay can be used for quantification of phospholipids in liposomes that are made in PBS or other phosphate based buffers.
2) The temperature of the heating block or the oven should be very closely controlled, so as not to exceed 200 degree centigrade, because then perchloric acid will evaporate resulting in a blue coloring of the samples at the final stage of the assay and also to not go below 180 centigrade to ensure total formation of inorganic phosphate.
3) Make sure that the standards and samples are treated in the same way and with the same solutions.