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Colorimetric estimation of phospholipids in aqueous dispersions
Journal of Biochemical and Biophysical Methods, 2 (1980) 251--255
251
© Elsevier/North-Holland Biomedical Press
COLORIMETRIC ESTIMATION OF PHOSPHOLIPIDS IN AQUEOUS DISPERSIONS
RAKESH MOHAN HALLEN
Department of Chemistry and The Biosystems Laboratories, Indian Institute of Technology, Kanpur 208016, U.P., India (Received 14 May 1979, accepted 22 November 1979)
A method for the estimation of phospholipids in aqueous dispersions is described. The method is based on the formation of a lipid--molybdenum blue complex, which is extracted into chloroform from the aqueous phase. Phosphate ions, detergents, proteins, neutral lipids and various other ions do not interfere in the lipid estimation. The method is sensitive down to a lipid concentration of 0.1 pmol/ml, with an accuracy better than -+3%. Key words: phospholipid vesicles; lipid--molybdenum blue complex; colorimetry.
INTRODUCTION
Phospholipid vesicles are currently recognised as good model systems for biological membranes [ 1 ]. In quantitative physico-chemical investigations of phospholipid vesicles, the concentration of the lipid is an important variable which needs to be determined accurately. Homogeneous phospholipid vesicle solutions of known concentrations cannot be prepared by dispersing a known quantity of the lipid, because the methods used for the preparation of the lipid vesicles usually yield a dispersion heterogeneous in vesicle size. Gel filtration [2] or ultracentrifugation [3] is then used to separate an almost homogeneous fraction. Both methods involve the loss of an indefinite fraction of the lipid originally dispersed. Lipid concentrations in such cases is c o m m o n l y determined by concentrated perchloric acid digestion of the lipid, followed by the determination o f phosphate ion concentration [4,5]. A dilute aqueous dispersion has, therefore, to be evaporated to dryness before digestion. In addition, if phosphate ions are present in the aqueous m e d i u m as buffer, t h e y will interfere in such a determination. London and Fergenson [6] have described a fluorimetric assay of aqueous phospholipid dispersions using the fluorescence e n h a n c e m e n t of diphenylhexatriene. However, the fluorescence e n h a n c e m e n t factor is a function of the concentrations of protein, neutral lipids, detergent and salts present in the dispersion. This m e t h o d , therefore, cannot be used without standardization for each set of conditions employed in an investigation. Raheja et al. [ 7 ] have described
252 a m e t h o d for the determination of the concentration of phospholipids using a m o l y b d e n u m reagent if the lipid is available as a chloroform solution. This m e t h o d is based on the formation of a chloroform-soluble phospholipid-m o l y b d e n u m blue complex [8]. In order to adapt this method for determination of phospholipid concentrations in aqueous dispersions, one may extract the lipids from the aqueous dispersions into chloroform and then use this method. However, this introduces errors in the estimation, because complete extraction is difficult. In this paper, we describe a m e t h o d similar to the method of Raheja et al. [7] to determine phospholipid concentrations in aqueous dispersions directly. We have further shown that this method is insensitive to the presence of some non-phospholipid solutes which may be present in vesicle solutions for physico~hemical studies. MATERIALS sn-3-Phosphatidylcholine (from hen's eggs), cardiolipin, lysolecithin, sphingomyelin, cholesterol and ceramides were obtained from C.S.I.R. Biochemical Center, Delhi. These lipids were chromatographically pure and were used as such. Phosphatidylserine was from I.C.N. Biochemicals, U.S.A. and was further purified on a silicic acid column. All other reagents were of analar quality. METHODOLOGY
Vesicle preparations Aqueous lipid dispersions were prepared either by a twenty-fold dilution of alcoholic solutions of the lipid with the desired aqueous medium or b y sonication. Molar lipid concentrations were determined b y phosphate analysis after digestion of the lipid with 70% perchloric acid [ 5].
Molybdenum reagent preparation 5 g of ammonium m o l y b d a t e is dissolved in 200 ml of 75% sulphuric acid (Solution A). 3 g o f ammonium m o l y b d a t e is dissolved in 50 ml of conc. HC1 and is then reduced b y magnetic stirring with 2 ml of mercury for a b o u t 30 min. This solution is then filtered and added slowly to Solution A. The dark-aquamarine coloured solution thus obtained is allowed to cool and is stored in a glass~stoppered bottle. The reagent is stable for at least 3 months at r o o m temperature.
Estimation To 2 ml of the phospholipid dispersion containing n o t more than 2/~mol of lipid in a glass-stoppered 120 × 12 mm test t u b e is added 1 ml of molybdenum reagent and the test t u b e is heated on a boiling water bath for a b o u t 2 min. The solution is allowed to cool for a b o u t 5 rain and 3.5 ml of a 9 : 1
253
chloroform/methanol mixture is added. The test t u b e is stoppered and the t w o phases mixed b y a slow rocking motion or on a cyclo mixer. The tube is allowed to stand for 20 min, when the t w o clear phases separate out. The lower blue chloroform phase is then removed either b y aspiration or with a separating funnel. The absorbance of this solution is read against a blank of pure chloroform at 760 nm, the absorbance maximum for the complex, in a 10 mm cuvette. RESULTS AND DISCUSSION
The minimum volume o f reagent required to give maximum absorbance after heating for 2 min is 0.4 ml per ml of the lipid dispersion (Fig. la). The minimum time for incubation of a mixture of 1.0 ml of reagent and 2.0 ml of dispersion at 100°C to obtain maximum absorbance is a b o u t 90 s (Fig. 15). Chloroform was chosen as a blank for convenience since the absorbance of a blank prepared with aqueous medium is equal to that of chloroform at 760 nm. The absorbance of the solution does n o t change for at least 40 min after the t w o phases separate out. The extinction coefficients at 760 nm and 30°C for the various lipids are given in Table 1. The absorbance is linear with the concentration of the lipids in the absorbance range 0.05--1.00. A typical plot of absorbance against concentration for sn-3-phosphatidylcholine is presented in Fig. 2. The effect of various solutes, which m a y be c o m m o n l y present in phospholipid vesicle preparations, on the estimation of sn-3-phosphatidylcholine is presented in Table 2. It is evident that there are no significant deviations. The conditions for the formation of the lipid--molybdenum complex used in this m e t h o d are expected to cause the oxidation of the phospholipids. No difference in the value of extinction coefficient while estimating an oxidized sample of phosphatidylcholine was observed. The major source of error is the failure to bring the complex to an equilibrium distribution between the t w o phases. It is, therefore, important that the TABLE 1 EXTINCTION COEFFICIENTS a BLUE COMPLEXES AT 30°C
OF
VARIOUS PHOSPHOLIPID--MOLYBDENUM
Lipid
Extinction coefficient (pmol-I . cm-1)
sn-3-Phosphatidylcholine
1.31 0.79 0.83 1,15 1.02
Lysophosphatidylcholine Phosphatidylserine Cardiolipin Sphingomyelin
+ 0.05 -+ 0.02 -+ 0.02 + 0.04 -+ 0.03
a A b s o r b a n c e / q u a n t i t y o f lipid (~mol), path length = 1 cm.
254
0.$
0.8 0.6 0.7 ~0.4
2
a -o 0.2
0
-
0.6
~ 0.5
~ 0.4
0.6
0.3
0.4
0.2
Ja 0.2
0.1
0
I
I
I
I
I
I
I
l
I
1 2 3 4 5 0 0.2 0.4 0.6 0.8 1.0 Time of incubation at 1()0°C ~lmotes of Lipid in rain Fig. 1. Absorbanee of chloroform phase as a function of (a) volume of reagent in ml used per ml of aqueous dispersion; (b) time of incubation (in rain) at IO0~C of the aqueous dispersion with the reagent. Fig. 2. Absorbance of chloroform phase as a function of the quantity of sn-3-phosphatidylcholine (in p m o l ; volume of CHCl 3 phase = 3.5 ml). TABLE 2 E F F E C T OF VARIOUS ADDITIONAL SOLUTES, IN THE DISPERSION, ON THE ESTIMATION OF sn-3-PHOSPHATIDYLCHOLINE Solvent
Absorbance a
Distilled water 5% ethanol + water 0.05 M CaCI2 0.10 M NaCI 0.5% sodium deoxycholate solution 0.01 M sodium phosphate 0.5% bovine serum albumin + 0.10 M NaCI 0.5% cholesterol + 0.10 M NaC1 0.5% Ceramide + 0.10 M NaCI
0.490 0.490 0.487 0.490 0.485 0.490 0.490 0.492 0.491
a Average o f t h r e e m e a s u r e m e n t s .
255
two phases be mixed as thoroughly as possible. However, vigorous mixing may sometimes lead to the formation of emulsions, that do not separate on standing. In such cases, separation can be achieved easily by centrifugation for 5--10 min. SHORT DESCRIPTION OF THE METHOD AND ITS ADVANTAGES A m o l y b d e n u m reagent is prepared by mixing a solution of ammonium molybdate in conc. HCI which has been reduced with mercury with a solution of ammonium molybdate in concentrated sulfuric acid. To 2 ml of phospholipid aqueous dispersion 1 ml of m o l y b d e n u m reagent is added and the mixture is heated for 2 min in a boiling water bath. It is then allowed to cool and a lipid--molybdenum blue complex is extracted with a 9 : 1 chloroform/methanol mixture. The absorbance at 760 nm of the blue chloroform solution is linear with the lipid concentration against a blank of pure chloroform. The method is sensitive down to a lipid concentration of 0.1 pmol per ml, with an accurary better than +3%. The method is more simple, rapid, selective and accurate than the methods used previously. Prior evaporation of dryness as required for P analysis is not necessary. The method of extracting the lipids from the aqueous dispersion into an organic solvent prior to colorimetric estimation with molybdenum reagent, leads to larger errors than this method. It is also more selective, i.e. the presence of other solutes (e.g. protein, detergents, neutral lipids, phosphate ions and calcium ions) do not affect the results. This selectivity makes it superior to the fluorimetric assay. ACKNOWLEDGEMENTS
The authors wishes to thank Dr. P. Gupta-Bhaya for encouraging discussions. Financial support from research grants to Dr. P. Gupta-Bhaya from C.S.I.R., New Delhi, and the Board of Nuclear Sciences, D.A.E., Bombay, is gratefully acknowledged. REFERENCES 1 Papahadjopolous, D. and Kimelberg, H.K. (1974) Prog. Surf. Sci. 4, 141--232 2 Huang, C. (1969) Biochemistry 8 , 3 4 4 - - 3 4 7 3 Barenholz, Y., Gibbes, D., Litman, B.J., Thompson, T.E., and Carlson, F.D. (1977) Biochemistry 16, 2806--2810 4 Fiske, C.K. and Subbarow, Y. (1925) J. Biol. Chem. 6 6 , 3 7 5 - - 4 0 0 5 Dittmer, J.C. and Wells, M.A. (1969) Methods Enzymol. 14, 384--487 6 London, E. and Fergenson, G.W. (1978) Anal. Biochem. 88, 203--211 7 Raheja, R.K., Kaur, C., Singh, A. and Bhatia, I.S. (1973) J. Lip. Res. 14,695--697 8 Galamo, D.S. (1970) Lipids 5, 573--575
251
© Elsevier/North-Holland Biomedical Press
COLORIMETRIC ESTIMATION OF PHOSPHOLIPIDS IN AQUEOUS DISPERSIONS
RAKESH MOHAN HALLEN
Department of Chemistry and The Biosystems Laboratories, Indian Institute of Technology, Kanpur 208016, U.P., India (Received 14 May 1979, accepted 22 November 1979)
A method for the estimation of phospholipids in aqueous dispersions is described. The method is based on the formation of a lipid--molybdenum blue complex, which is extracted into chloroform from the aqueous phase. Phosphate ions, detergents, proteins, neutral lipids and various other ions do not interfere in the lipid estimation. The method is sensitive down to a lipid concentration of 0.1 pmol/ml, with an accuracy better than -+3%. Key words: phospholipid vesicles; lipid--molybdenum blue complex; colorimetry.
INTRODUCTION
Phospholipid vesicles are currently recognised as good model systems for biological membranes [ 1 ]. In quantitative physico-chemical investigations of phospholipid vesicles, the concentration of the lipid is an important variable which needs to be determined accurately. Homogeneous phospholipid vesicle solutions of known concentrations cannot be prepared by dispersing a known quantity of the lipid, because the methods used for the preparation of the lipid vesicles usually yield a dispersion heterogeneous in vesicle size. Gel filtration [2] or ultracentrifugation [3] is then used to separate an almost homogeneous fraction. Both methods involve the loss of an indefinite fraction of the lipid originally dispersed. Lipid concentrations in such cases is c o m m o n l y determined by concentrated perchloric acid digestion of the lipid, followed by the determination o f phosphate ion concentration [4,5]. A dilute aqueous dispersion has, therefore, to be evaporated to dryness before digestion. In addition, if phosphate ions are present in the aqueous m e d i u m as buffer, t h e y will interfere in such a determination. London and Fergenson [6] have described a fluorimetric assay of aqueous phospholipid dispersions using the fluorescence e n h a n c e m e n t of diphenylhexatriene. However, the fluorescence e n h a n c e m e n t factor is a function of the concentrations of protein, neutral lipids, detergent and salts present in the dispersion. This m e t h o d , therefore, cannot be used without standardization for each set of conditions employed in an investigation. Raheja et al. [ 7 ] have described
252 a m e t h o d for the determination of the concentration of phospholipids using a m o l y b d e n u m reagent if the lipid is available as a chloroform solution. This m e t h o d is based on the formation of a chloroform-soluble phospholipid-m o l y b d e n u m blue complex [8]. In order to adapt this method for determination of phospholipid concentrations in aqueous dispersions, one may extract the lipids from the aqueous dispersions into chloroform and then use this method. However, this introduces errors in the estimation, because complete extraction is difficult. In this paper, we describe a m e t h o d similar to the method of Raheja et al. [7] to determine phospholipid concentrations in aqueous dispersions directly. We have further shown that this method is insensitive to the presence of some non-phospholipid solutes which may be present in vesicle solutions for physico~hemical studies. MATERIALS sn-3-Phosphatidylcholine (from hen's eggs), cardiolipin, lysolecithin, sphingomyelin, cholesterol and ceramides were obtained from C.S.I.R. Biochemical Center, Delhi. These lipids were chromatographically pure and were used as such. Phosphatidylserine was from I.C.N. Biochemicals, U.S.A. and was further purified on a silicic acid column. All other reagents were of analar quality. METHODOLOGY
Vesicle preparations Aqueous lipid dispersions were prepared either by a twenty-fold dilution of alcoholic solutions of the lipid with the desired aqueous medium or b y sonication. Molar lipid concentrations were determined b y phosphate analysis after digestion of the lipid with 70% perchloric acid [ 5].
Molybdenum reagent preparation 5 g of ammonium m o l y b d a t e is dissolved in 200 ml of 75% sulphuric acid (Solution A). 3 g o f ammonium m o l y b d a t e is dissolved in 50 ml of conc. HC1 and is then reduced b y magnetic stirring with 2 ml of mercury for a b o u t 30 min. This solution is then filtered and added slowly to Solution A. The dark-aquamarine coloured solution thus obtained is allowed to cool and is stored in a glass~stoppered bottle. The reagent is stable for at least 3 months at r o o m temperature.
Estimation To 2 ml of the phospholipid dispersion containing n o t more than 2/~mol of lipid in a glass-stoppered 120 × 12 mm test t u b e is added 1 ml of molybdenum reagent and the test t u b e is heated on a boiling water bath for a b o u t 2 min. The solution is allowed to cool for a b o u t 5 rain and 3.5 ml of a 9 : 1
253
chloroform/methanol mixture is added. The test t u b e is stoppered and the t w o phases mixed b y a slow rocking motion or on a cyclo mixer. The tube is allowed to stand for 20 min, when the t w o clear phases separate out. The lower blue chloroform phase is then removed either b y aspiration or with a separating funnel. The absorbance of this solution is read against a blank of pure chloroform at 760 nm, the absorbance maximum for the complex, in a 10 mm cuvette. RESULTS AND DISCUSSION
The minimum volume o f reagent required to give maximum absorbance after heating for 2 min is 0.4 ml per ml of the lipid dispersion (Fig. la). The minimum time for incubation of a mixture of 1.0 ml of reagent and 2.0 ml of dispersion at 100°C to obtain maximum absorbance is a b o u t 90 s (Fig. 15). Chloroform was chosen as a blank for convenience since the absorbance of a blank prepared with aqueous medium is equal to that of chloroform at 760 nm. The absorbance of the solution does n o t change for at least 40 min after the t w o phases separate out. The extinction coefficients at 760 nm and 30°C for the various lipids are given in Table 1. The absorbance is linear with the concentration of the lipids in the absorbance range 0.05--1.00. A typical plot of absorbance against concentration for sn-3-phosphatidylcholine is presented in Fig. 2. The effect of various solutes, which m a y be c o m m o n l y present in phospholipid vesicle preparations, on the estimation of sn-3-phosphatidylcholine is presented in Table 2. It is evident that there are no significant deviations. The conditions for the formation of the lipid--molybdenum complex used in this m e t h o d are expected to cause the oxidation of the phospholipids. No difference in the value of extinction coefficient while estimating an oxidized sample of phosphatidylcholine was observed. The major source of error is the failure to bring the complex to an equilibrium distribution between the t w o phases. It is, therefore, important that the TABLE 1 EXTINCTION COEFFICIENTS a BLUE COMPLEXES AT 30°C
OF
VARIOUS PHOSPHOLIPID--MOLYBDENUM
Lipid
Extinction coefficient (pmol-I . cm-1)
sn-3-Phosphatidylcholine
1.31 0.79 0.83 1,15 1.02
Lysophosphatidylcholine Phosphatidylserine Cardiolipin Sphingomyelin
+ 0.05 -+ 0.02 -+ 0.02 + 0.04 -+ 0.03
a A b s o r b a n c e / q u a n t i t y o f lipid (~mol), path length = 1 cm.
254
0.$
0.8 0.6 0.7 ~0.4
2
a -o 0.2
0
-
0.6
~ 0.5
~ 0.4
0.6
0.3
0.4
0.2
Ja 0.2
0.1
0
I
I
I
I
I
I
I
l
I
1 2 3 4 5 0 0.2 0.4 0.6 0.8 1.0 Time of incubation at 1()0°C ~lmotes of Lipid in rain Fig. 1. Absorbanee of chloroform phase as a function of (a) volume of reagent in ml used per ml of aqueous dispersion; (b) time of incubation (in rain) at IO0~C of the aqueous dispersion with the reagent. Fig. 2. Absorbance of chloroform phase as a function of the quantity of sn-3-phosphatidylcholine (in p m o l ; volume of CHCl 3 phase = 3.5 ml). TABLE 2 E F F E C T OF VARIOUS ADDITIONAL SOLUTES, IN THE DISPERSION, ON THE ESTIMATION OF sn-3-PHOSPHATIDYLCHOLINE Solvent
Absorbance a
Distilled water 5% ethanol + water 0.05 M CaCI2 0.10 M NaCI 0.5% sodium deoxycholate solution 0.01 M sodium phosphate 0.5% bovine serum albumin + 0.10 M NaCI 0.5% cholesterol + 0.10 M NaC1 0.5% Ceramide + 0.10 M NaCI
0.490 0.490 0.487 0.490 0.485 0.490 0.490 0.492 0.491
a Average o f t h r e e m e a s u r e m e n t s .
255
two phases be mixed as thoroughly as possible. However, vigorous mixing may sometimes lead to the formation of emulsions, that do not separate on standing. In such cases, separation can be achieved easily by centrifugation for 5--10 min. SHORT DESCRIPTION OF THE METHOD AND ITS ADVANTAGES A m o l y b d e n u m reagent is prepared by mixing a solution of ammonium molybdate in conc. HCI which has been reduced with mercury with a solution of ammonium molybdate in concentrated sulfuric acid. To 2 ml of phospholipid aqueous dispersion 1 ml of m o l y b d e n u m reagent is added and the mixture is heated for 2 min in a boiling water bath. It is then allowed to cool and a lipid--molybdenum blue complex is extracted with a 9 : 1 chloroform/methanol mixture. The absorbance at 760 nm of the blue chloroform solution is linear with the lipid concentration against a blank of pure chloroform. The method is sensitive down to a lipid concentration of 0.1 pmol per ml, with an accurary better than +3%. The method is more simple, rapid, selective and accurate than the methods used previously. Prior evaporation of dryness as required for P analysis is not necessary. The method of extracting the lipids from the aqueous dispersion into an organic solvent prior to colorimetric estimation with molybdenum reagent, leads to larger errors than this method. It is also more selective, i.e. the presence of other solutes (e.g. protein, detergents, neutral lipids, phosphate ions and calcium ions) do not affect the results. This selectivity makes it superior to the fluorimetric assay. ACKNOWLEDGEMENTS
The authors wishes to thank Dr. P. Gupta-Bhaya for encouraging discussions. Financial support from research grants to Dr. P. Gupta-Bhaya from C.S.I.R., New Delhi, and the Board of Nuclear Sciences, D.A.E., Bombay, is gratefully acknowledged. REFERENCES 1 Papahadjopolous, D. and Kimelberg, H.K. (1974) Prog. Surf. Sci. 4, 141--232 2 Huang, C. (1969) Biochemistry 8 , 3 4 4 - - 3 4 7 3 Barenholz, Y., Gibbes, D., Litman, B.J., Thompson, T.E., and Carlson, F.D. (1977) Biochemistry 16, 2806--2810 4 Fiske, C.K. and Subbarow, Y. (1925) J. Biol. Chem. 6 6 , 3 7 5 - - 4 0 0 5 Dittmer, J.C. and Wells, M.A. (1969) Methods Enzymol. 14, 384--487 6 London, E. and Fergenson, G.W. (1978) Anal. Biochem. 88, 203--211 7 Raheja, R.K., Kaur, C., Singh, A. and Bhatia, I.S. (1973) J. Lip. Res. 14,695--697 8 Galamo, D.S. (1970) Lipids 5, 573--575