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Gravimetric determination of phospholipid concentration
Chemistry and Physics of Lipids 165 (2012) 689–695
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Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip
Gravimetric determination of phospholipid concentration Roberto Tejera-Garcia a , Lisa Connell b , Walter A. Shaw b , Paavo K.J. Kinnunen a,∗ a Helsinki Biophysics and Biomembrane Group, Department of Biomedical Engineering and Computational Science, School of Sciences, Aalto University, P.O. Box 12200, FI-00076 Espoo, Finland b Avanti Polar Lipids, Inc., Alabaster, AL 35007, United States
a r t i c l e
i n f o
Article history: Received 23 April 2012 Received in revised form 6 June 2012 Accepted 18 June 2012 Available online 5 July 2012 Keywords: Phospholipid concentration Phosphate assay Microbalance Gravimetric analysis
a b s t r a c t Accurate determination of lipid concentrations is an obligatory routine in a research laboratory engaged in studies using this class of biomaterials. For phospholipids, this is frequently accomplished using the phosphate assay (Bartlett, G.R. Phosphorus Assay in Column Chromatography. J. Biol. Chem. 234, 466–468, 1959). Given the purity of the currently commercially available synthetic and isolated natural lipids, we have observed that determination of the dry weight of lipid stock solutions provides the fastest, most accurate, and generic method to assay their concentrations. The protocol described here takes advantage of the high resolution and accuracy obtained by modern weighing technology. We assayed by this technique the concentrations of a number of phosphatidylcholine samples, with different degrees of acyl chain saturation and length, and in different organic solvents. The results were compared with those from Bartlett assay, 31 P NMR, and Langmuir compression isotherms. The data obtained show that the gravimetric assay yields lipid concentrations with a resolution similar or better than obtained by the other techniques. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Accurate determination of lipid stock solution concentrations is mandatory in both basic as well as applied studies on lipids. More specifically, the biophysical properties of lipid monolayers, supported lipid bilayers, and liposomes are in most cases profoundly influenced by even minor variations in the molar ratios of multicomponent lipid mixtures (Tejera-Garcia et al., 2011). As an example, the drug release characteristics of thermosensitive liposome formulations are critically dependent on even minor differences in the relative contents of their constituent lipids, reflecting the sensitivity of lipid membrane thermal phase behavior to the exact stoichiometry of its components (Banno et al., 2010; Wang and Thanou, 2010). Even when rigorously handled according
Abbreviations: DPPC, 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine; C, in chloroform; E, in ethanol; X, in a chloroform, methanol, water mixture (65:35:8, by volume); Di-18:2 PC, 1,2-di-(9Z,12Z-octadecadienoyl)-sn-glycero-3phosphocholine; A, total monolayer area; LC, liquid condensed phase; LE, liquid expanded phase; N, number of molecules; 31 P NMR, phosphorus-31 nuclear magnetic resonance; V, volume; , surface pressure. ∗ Corresponding author at: Helsinki Biophysics and Biomembrane Group (HBBG), Department of Biomedical Engineering and Computational Science, Aalto University, Otakaari 3J, 3rd floor, 02150 Espoo, Finland. Tel.: +358 5 05404600; fax: +358 9 470 23182. E-mail address: paavo.kinnunen@aalto.fi (P.K.J. Kinnunen). 0009-3084/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemphyslip.2012.06.005
to the lipid manipulation directives (Moore et al., 2007), the volatility of commonly used lipid solvents such as chloroform necessitates frequent routine control of lipid stock solution concentrations. The development of a universal method for the accurate determination of lipid stock solution concentrations is not straightforward due to the chemical diversity of lipid species (Fahy et al., 2005), and because of the inherent difficulties in the preparation of pure lipid standards of stable concentration. Protocols used today for the assessment of lipid stock solution concentrations are commonly based on techniques originally developed for the characterization of more complex systems. In 1925, Fiske and Subbarow designed a phosphate assay for the analysis of urine and blood samples, employing strong acids to liberate and oxidize phosphorus in the sample, producing phosphate ions that in the presence of proper reducing reagents form colored complexes amenable to quantification by absorbance spectroscopy (Fiske and Subbarow, 1925). This assay was subsequently adapted for the analysis of a variety of different samples and improved to obtain higher sensitivity. Along these lines, the method of Bartlett is at present perhaps the most widely employed in phospholipid research (Bartlett, 1959). Due to its reproducibility, requiring no expensive reagents, and being rather fast to complete (about 3 h), the Bartlett assay can be considered as a reference procedure for assaying phospholipid concentration (Söderlund et al., 1997; Fenske and Cullis, 2005). A complete description of a modern variant of this assay can be found on the Avanti Polar
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Lipids website (http://www.avantilipids.com - Determination of Total Phosphorus). Nevertheless, this assay involves some inconveniences: i. It is time and resource consuming, requiring strict cleaning protocols, usually involving acid washing, to keep the glassware used phosphate free. A continuous supply of fresh reagents is needed to obtain consistent results, especially in laboratories where it is run as a routine. The preparation of the necessary standard curves is time consuming. ii. Most of the assay must be performed using fume hoods and protective equipment, like gloves and glasses, because of the inherent risks of handling strong acids at high temperatures. The explosion hazard associated with possible complete evaporation of the sample is controlled using glass marbles covering the tubes and constantly running water flows on the fume hood faucets to increase humidity. 31 P
NMR measures the electromagnetic energy emitted during the reorientation of the spin polarization of 31 P isotopes, in the presence of an external magnetic field, after perturbation at controlled radiofrequency. Measurement of lipid stock solution concentrations by this technique has been described (http://www.avantilipids.com - Phospholipid Analysis, 31P NMR). 31 P NMR also allows the assay of phospholipid concentration without separation or other kind of sample manipulation and even quantification of individual phospholipids in multi-component mixtures (Sotirhos et al., 1986). It produces more precise and accurate data than the Bartlett assay and has been recently refined for use with liposomes (Dubinnyi et al., 2006). However, its use for routine measurement of lipid stock solutions is limited by instrument availability and due to large amounts of material required. Several other techniques have been employed for the measurement of lipid stock solution concentrations although none of them has reached the popularity of the Bartlett phosphate assay. These include inductively coupled plasma-mass spectrometry (ICP-MS) (Moore et al., 2007; de Smet et al., 2010); and separation based techniques such as evaporative light-scattering detection (ELSD), and refractometric and spectrophotometric detectors (Christie, 1992). The limitations inherent to most of the above techniques, together with the development of affordable and precise microbalances, prompted us to develop gravimetric analysis for the measurement of lipid stock solution concentrations (Zhao et al., 2005), described here in detail. Its simplicity and precision make this assay superior to the other methods. The possibility of eventually recovering the pure dry lipid after the measurement, as well as no need for specific and in some cases expensive laboratory equipment, explicit reagents or previous user expertise, are some of the inherent advantages of this method. The evaluation of the protocol described here shows that the use of gravimetric assay performed with a modern high precision microbalance, allows to obtain data comparable or better than obtained by some of the most precise techniques available today. Our assay is more accurate than the traditional methods, which are now routinely used in most laboratories.
2. Materials 1,2-Dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-di-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine (di -18:2 PC) in the indicated solvents, C: chloroform, E: in ethanol, and X: in a chloroform, methanol, water mixture (65:35:8, by volume), were from Avanti Polar Lipids (Alabaster, AL). Other chemicals were of analytical grade and from standard sources.
3. Experimental 3.1. Bartlett phosphorus assay Solutions of DPPC and di-18:2 PC in solvents C, E, and X, each at concentrations of 2.0, 0.8, and 0.4 mg/mL were prepared for analysis by the Barlett phosphorus method as described (http://www.avantilipids.com - Determination of Total Phosphorus). Approximately 0.1 mol of each sample was added to the bottom of respective reaction tubes in triplicate using a glass gastight Hamilton syringe (Hamilton Company Reno NV). Solutions were evaporated to dryness at room temperature with a gentle flow of grade 5 nitrogen gas. Standards of 0.0325–0.2280 mol were prepared in like manner by delivering appropriate volumes of 0.65 mM stock solution (Sigma, St. Louis, MO). After reaction, the solutions were assayed on an Agilent (Englewood, CA) model 8453 diode array spectrophotometer scanning from 190–850 nm. Phosphorus concentrations were calculated by reading the absorbance intensity at 820 nm plotted from the linear standard curve and converted to mg/mL and mM concentrations using the molecular weights of 734.04 g/mol for DPPC and 782.08 g/mol for di-18:2 PC. Precision for triplicate assay was <2%. 3.2.
31 P-nuclear
magnetic resonance
The DPPC and di-18:2 PC solutions described above were assayed by 31 P NMR after transferring 0.5 mL (0.25–1.5 mol) to 1.0 mL volumetric tubes with a glass gas tight syringe. These solutions were dried to a residue using a gentle stream of grade 5 nitrogen at room temperature. The residues were dissolved in 1 mL of sodium cholate detergent. Solutions were assayed on a Bruker AvanceTM 400 MHz NMR (Bruker Daltonik, Bremen, Germany) system calibrated with an external standard of dioleoyl phosphatidylcholine (18:1 PC) (Avanti Polar Lipids, Alabaster AL). Quality control samples of dioleoyl phosphatidylethanolamine assayed with 100 ± 2% accuracy. 3.3. Gravimetric assay In this study the microbalance and the set of accessories employed in the experiments described were contained in the SuperG lipid gravimetric analysis kit (Kibron Inc., Helsinki, Finland). In addition to the microbalance, the kit includes a set of aluminum sample pans for weighing (max. capacity, 80 l), a pair of inverse clamp curved tweezers for pan handling, avoiding contact with the internal surface of the sample pans, and a storage box with labeled separate compartments for sample identification. The assay is performed as a series of default steps following the instructions of the microbalance’s software: 1. The needed number of pans was selected and placed each into the assigned numbered compartments according to the number of samples to be measured. 2. The tare masses of the empty pans were measured and recorded in a log file of the software supervising the assay. After each measurement the pans were returned to their respective compartment in the box. 3. Next, boxes containing the empty pans were placed under a moderate stream of nitrogen and a specified volume (from 10 to 80 l) of lipid sample was deposited drop by drop in each pan using a microsyringe (Hamilton, Bonaduz, Switzerland). Subsequently, the samples were maintained under nitrogen flow until complete evaporation of the liquid phase, taking approximately 5–30 min, depending on the lipid type, solution volume, and nitrogen flow rate. As an alternative, and in order to prevent oxidation especially when working with polyunsaturated
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lipids, this procedure was also performed inside a nitrogen saturated atmosphere without the need of a continuous nitrogen flow in the process (see Recording Langmuir-film compression isotherms below). 4. Once the evaporation was complete, the samples were placed under reduced pressure (∼4 kPa) to remove possible traces of solvent for approximately 2 h. Comparison of the mass measured at different times allows to estimate the optimum time at reduced pressure for removal of solvent traces. 5. Finally, the masses of the different pans containing the dry lipid residues were measured by the microbalance, with the dry lipid net mass obtained after subtraction of the respective empty pan tare weight. The software then calculated the masses and molar concentrations of the lipids, according to the volumes and molecular weights provided by the user and stored in the database of the software. The weight of the hydration water on the lipid molecules headgroups was automatically subtracted from the lipid mass in all the experiments. The subtraction of this mass is important especially when comparing concentrations measured gravimetrically with those obtained by techniques measuring the amount of anhydrous phosphorus, such as NMR or phosphate assay. The precise percentage (weight) of water of hydration present in a lipid sample is usually obtained from the lipid manufacturer. The specific value of the water of hydration for the phosphatidylcholines employed here was specified as 4.4% of the total weight, which corresponds to approximately two molecules of water per head group. In cases where precise value is not available, approximate percentage of the water of hydration can be found in literature (Jendrasiak and Hasty, 1974; Jendrasiak et al., 1996). The error introduced is usually of the order of ±1 water molecule which can decrease the precision of the concentrations determined gravimetrically. In the present study this decrease in precision would represent an extra 1% in the absolute error associated to the concentrations determined 3.4. Recording Langmuir-film compression isotherms As an independent technique we additionally assessed the concentrations of the samples from compression isotherms (surface pressure, , vs. monolayer area, A) of lipid monolayers recorded at a gas/water interface. The measurement of was performed as ( 0 – ), 0 and being the interfacial tensions in the absence and in the presence of the lipid monolayer, respectively. In this study all compression isotherms ( vs. A) were recorded using a Langmuir trough (MTXL, Kibron Inc., Helsinki, Finland) equipped with a microbalance and Wilhelmy-Padday rod probe (Padday et al., 1975). The aqueous subphase solution was 40 ml of 10 mM KH2 PO4 , 100 mM NaCl in deionized filtered water (Direct-Q UV; Millipore, Jaffrey, NH), yielding a total surface area of 228 cm2 . This buffer makes the phospholipid hydration shells uniform (Sabatini et al., 2006) and was prepared daily prior every set of experiments and kept at 4 ◦ C. All measurements were performed at room temperature (24 ◦ C), inside a controlled atmosphere chamber (850 Series, Plas-Labs Inc., Lansing, MI, USA) saturated with nitrogen to avoid oxidation when working with polyunsaturated phospholipids. Saturation was achieved by keeping a constant nitrogen flow rate, while discharging the chamber at the same flow using the chamber’s vacuum pump. Complete saturation was considered after recording levels of oxygen below 2% with a dissolved oxygen meter (ISO2 ; WPI, Stevenage, UK). The chamber’s taps were closed and the experiments were performed with continuous monitoring of the stability of the oxygen levels. In this nitrogen atmosphere, a microsyringe (Hamilton, Bonaduz, Switzerland) was filled with the indicated amount of the stock solution and the main stock was hermetically closed
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to diminish possible evaporation. At this point, part of the volume of original solution in the microsyringe was separated and stored for later assay by gravimetric analysis. The sample in the microsyringe was carefully deposited onto the gas/water interface in the trough, whereafter the monolayer was allowed to stabilize for 10 min for complete evaporation of the solvents. Subsequently, two symmetrically moving Teflon barriers were used to compress the lipid monolayer at a rate of 3.6 cm2 /min, allowing reorientation and relaxation of the lipids during compression. The amount of lipid solution deposited for each sample was estimated by an initial coarse measurement of dry lipid content after evaporation in air over a hot plate (50 ◦ C). All experiments were performed twice using two aliquots of the same sample. If differences above 1 mN/m, 2 A˚ 2 /molecule, or 20 mV were found a third isotherm was recorded. For DPPC, the values of A corresponding to the cross-sectional limiting area, obtained from the extrapolation to zero surface pressure of the linear part of the compression isotherm at surface pressures around 30 mN/m, were employed for the calculation of the number of molecules (N) in the monolayer, after dividing this value by the respective areas per molecule obtained from data in the literature (50 A˚ 2 per DPPC molecule when extrapolating from data at surface pressures around 30 mN/m – see Fig. S1, panel A, top) (Smaby et al., 1996). The number of molecules derived was then employed to calculate the concentration of the original solution by dividing N by the volume (V) of the liquid solution applied onto the interface. For validation of the lipid concentration obtained, the area per molecule corresponding to the onset of the liquid expanded (LE) to liquid condensed (LC) phase transition was obtained from the recorded compression isotherms and compared with the corresponding data in the literature (71 A˚ 2 per DPPC molecule – see Fig. S1, panel A). These areas were identified as changes from positive to negative in the values of the third derivative of the vs. A function, as described by Brockman et al. (1980). For di-18:2 PC, the lipid concentrations were obtained as N/V. However, di-18:2 PC does not form LC or solid phases under the experimental conditions used. Therefore, it was not possible to use the cross-sectional limiting area or critical points of the isotherms for the calculation of N. Instead, N was obtained by fitting an adjustable parameter in the following monolayer equation of state for the LE region (Wolfe and Brockman, 1988): =
qkT ln ω1
1 f
1+
ω1 A/N − ω0
(1)
where k is the Boltzmann constant, T is temperature, ω0 the area of dehydrated lipid ( = ∞), ω1 = 9.65 A˚ 2 is the surface area occupied by one water molecule at the interface, and q and f are the activity coefficients of lipid and water, respectively. The values for di-18:2 PC of ω0 , f, and q, obtained from data in the literature (Smaby et al., 1997), were 44.86, 1.13, and 3.46 A˚ 2 , respectively. This value of ω0 was introduced in Eq. (1) and the values of f and q, obtained as fitting parameters for the isotherms recorded, and compared with the values in the literature for validation. Approximate correlation between q and f (ln q > 7.1 − 5.2f) has been proven for pure lipids (Smaby and Brockman, 1991). We consider this correlation as additional support on the accuracy of the obtained values of N. 4. Results 4.1. Characteristics of the SuperG microbalance The SuperG microbalance is routinely calibrated using an OIML class E1 standard weight of 1 mg (KERN & Sohn GmbH, Balingen, Germany) prior to its daily use, following the instructions provided by the instrument’s software. The precision (repeatability) of the
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Fig. 1. Standard deviations for ten readings of up to six standard 1 mg weights. Linearity of the balance is depicted in the inset.
instrument is provided by the manufacturer and is given according to respective sensitivity stability values. However, these values are approximate only and in order to exclude possible malfunction and to have confidence on the accuracy and precision of the dry weights measured, it is recommended to evaluate these parameters when working with an instrument in intensive use. The precision of the microbalance was evaluated as the standard deviation for ten separate measurements of a reference pan loaded with up to six 1 mg standard weights. Below 2 mg, the microbalance’s precision is about ±0.2 g (Fig. 1). Above this mass the precision decreases with the increase in load going from ±1.5 g for samples of around 3 mg up to ±2.5 g for samples of around 6 mg. The accuracy of the microbalance measurements is guaranteed as 100%, within the error interval given by the instrument’s precision, by the use of certified measurement standards. For the gravimetric assay the masses of the standard aluminum pans were first recorded. The same pans were then used to apply known volumes of the lipid stock solutions and for recording the dry weight of the lipids after evaporation of the solvents. In this way the net mass of dry lipid was obtained for each sample. 4.2. Bartlett’s assay and 31 P NMR Comparison of the lipid concentrations measured for the different solutions using the Bartlett and 31 P NMR methods reveals minor differences between the concentrations measured by these methods (Fig. 4). For most measurements 31 P NMR yields a higher precision with smaller error than the Bartlett assay.
5346 ± 6 mm2 for DPPC monolayers spread from C: chloroform, E: ethanol, and X: chloroform, methanol, water mixture (65:35:8, by volume), respectively. Using a limiting cross-sectional area of DPPC of 50 ± 1 A˚ 2 per molecule, these measured areas correspond to 18. 7 ± 0.4, 19.2 ± 0.4, and 17.8 ± 0.4 nmoles of DPPC, for the monolayers spread from the above solvents [19]. The original molar lipid concentration on each solution could therefore be determined (see Supplementary Material) as the ratio between the calculated monolayer lipid content and the corresponding volume of solution employed for monolayer deposition, yielding 2.67 ± 0.09, 0.51 ± 0.02, and 1.11 ± 0.02 mM for DPPC in C, E, and X, respectively (Fig. 4). As a further control for the calculated amounts of DPPC in the Langmuir films, the area per molecule corresponding to the onset of the LE/LC phase transition was obtained as described above, corresponding to monolayer areas of 8033 ± 6, 7967 ± 6, and 7477 ± 6 mm2 for films spread from C, E, and X, respectively (Fig. 2, top row, blue dashed line). For the DPPC concentrations calculated above, these areas correspond to 71.3 ± 1.6, 68.9 ± 1.5, and 69.8 ± 1.6 A˚ 2 per molecule, which all correspond within error to the data in the literature, 71 ± 1 A˚ 2 per molecule [19], thus confirming the accuracy of the measured lipid concentrations. Compression isotherms recorded for the di-18:2 PC monolayers exhibit the gas and LE phases at the experimental conditions used (Fig. 3). By introducing into Eq. (1) the value of ω0 = 44.86 A˚ 2 calculated from the data in the literature [22] and fitting the recorded isotherms, we obtained the corresponding values for f, q, and N. The number of molecules obtained were N = 154 × 1014 , 118 × 1014 , and 180 × 1014 for the di-18:2 PC monolayers spread from C, E, and X, respectively. Taking into account the sample volumes applied onto the gas/water interface in each case, the molar lipid concentrations could be determined as the ratio between the calculated monolayer lipid contents and these volumes of applied solution, yielding 2.56 ± 0.03, 0.98 ± 0.01, and 0.5 ± 0.01 mM for di-18:2 PC in C, E, and X, respectively (Fig. 4). The values for the activity coefficients of lipid and water obtained after fitting were f = 1.13, 1.12, and 1.13; and q = 2.97, 3.68, and 3.33 for di-18:2 PC in C, E, and X, respectively. These values are in reasonable agreement with those obtained from data in the literature [22]: f = 1.13 and q = 3.46, thus confirming the accuracy of the concentrations obtained. This agreement is clearly better for the f values than for q, maybe due to the higher exposure of the water interfacial activity to minor environmental perturbations than the one of the lipid. Nevertheless, the theoretical correlation for pure systems between these two parameters (Ln q > 7.1 − 5.2f) holds quite accurately with the solutions studied, with relative errors below 15% between the values of q obtained by this relation and the ones obtained directly as fitting parameters, thus validating the accuracy of the di-18:2 PC concentrations obtained by this method.
4.3. Langmuir-film compression isotherms An independent check on the phospholipid concentrations was obtained from the analysis of the compression isotherms of lipid monolayers prepared from the indicated lipid solutions. The determination of the lipid concentrations from the recorded compression isotherms was calculated as the number of lipid molecules per monolayer divided by V, as described above. Compression isotherms recorded for DPPC monolayers prepared from different solvents exhibit the typical distinct phases appearing during compression (Fig. 2), viz.: gaseous, liquid expanded, and liquid condensed. The plateau, corresponding to the LE/LC two-phase region appears at surface pressures of ∼9 mN/m as described in the literature [19]. The values of A corresponding to the DPPC cross-sectional limiting area are indicated by the red dotted lines in Fig. 2. These areas are 5621 ± 6, 5788 ± 6, and
4.4. Gravimetric assay The lipid dry weight obtained after the deposition of 80 ± 1 l of each solution and the subsequent removal of the solvent as described above are summarized in Table 1. After dividing the
Table 1 Mass of dry lipid after solvent removal from the solutions DPPC in C, DPPC in E, DPPC in X, di-18:2 PC in C, di-18:2 PC in E, and di-18:2 PC in X.
DPPC (g) di-18:2 PC (g)
C
E
X
2.07 ± 0.08 0.41 ± 0.01
1.84 ± 0.02 0.34 ± 0.03
1.94 ± 0.03 0.37 ± 0.01
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Fig. 2. Langmuir compression isotherms, and their consecutive derivatives, for DPPC monolayers spread from (left column) chloroform, (middle) ethanol, and (right) chloroform, methanol, water mixture (65:35:8, by volume). The red dotted line indicates the limiting area for the DPPC molecule and the blue dashed line specifies the areas for the onset of the LE/LC phase transition. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
ratio between these masses and the original volume of solution by the correspondent molecular weights the respective molar lipid concentrations were as compiled in Table S1 in Supplementary material (see also Fig. 4). These concentrations were within the
concentration limits of the gravimetric assay. The maximum and minimum concentration limits as well as the errors in the concentrations determined by gravimetric assay depend on the precision of the microbalance and the microsyringe resolution, the lipid in
Fig. 3. Langmuir compression isotherms for di-18:2 PC monolayers spread from (dotted line, middle) chloroform, (solid line, left) ethanol, and (dashed line, right) chloroform, methanol, water mixture (65:35:8, by volume).
Fig. 4. Concentrations of the DPPC and di-18:2 PC solutions in C: chloroform, E: ethanol, and X: in chloroform, methanol, water (65:35:8 by volume), obtained from Bartlett’s phosphate assay, 31 P NMR, Langmuir compression isotherms and gravimetric assay.
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question and its concentration, and the volume of the sample analyzed. The computation of these limits has been implemented in the software of the microbalance used in this study, specifically designed for gravimetric analysis. The minimum concentration accessible to gravimetric analysis is limited by the minimum weight measurable by the microbalance. For SuperG this minimum weight is 1 g, which when applied in the maximum volume of sample applicable (80 l) corresponds to 1.36 and 1.28 nM for DPPC and di-18:2 PC, respectively. Depending on the assay parameters, maximum concentration limit is determined by the precision of the volumes deposited (± 0.5 l for the microsyringe employed here), which causes the accuracy of the assay to decrease at higher lipid concentrations. Results from the concentrations determined using the Bartlett assay and monolayer studies present absolute errors of up to 0.1 mM (Fig. 4 and Table S1). For DPPC and di-18:2 PC the maximum concentration limit would correspond to approximately 15.5 mM.
5. Discussion Comparison of the lipid concentrations of the solutions in different solvents measured by the methods used in this study reveal similar concentration values for each solution, in most cases with overlapping error intervals. The set of stock solutions studied shows no systematic deviation for any of the techniques in the concentration values obtained and only minor differences are evident between the different techniques in some singular cases. For DPPC solutions the concentrations obtained by the Bartlett method are slightly higher than the ones from other techniques, while the ones from 31 P NMR are lower. This is likely to be due to evaporation of the solvents which is an inherent problem when working with organic solvent solutions. In this particular case, evaporation as a source of extra error could be due to the fact that, even when same original solutions were employed for both measurements, technical limitations required the use of different ampoules for the Bartlett and 31 P NMR assays. Concentrations obtained from compression isotherms and gravimetric assay were studied simultaneously and from the same ampoule, and do show closely parallel values thus strengthening the above line of reasoning. Concentrations obtained by the gravimetric assay represent values intermediate to those measured by the other methods, yet with similar or smaller errors. 31 P NMR and Langmuir compression isotherms were both precise for the determination of lipid stock solution concentrations. However, as mentioned above, from a practical point of view the application of these techniques for routine evaluation of lipid stocks in a laboratory is not feasible. The relevance of the Bartlett method and other methods based on the determination of phosphorus contents in lipid extraction and separation processes is practical. However, for routine measurement of lipid stock solution concentrations, the time and resources that this technique require makes the gravimetric assay attractive. The microbalance employed in this study fulfilled perfectly the sensitivity requirements of the gravimetric assay. We consider this assay to represent an optimum routine tool in any laboratory for the evaluation of lipid stock solution concentrations. Accurate gravimetric determination of phospholipid concentrations in stock solutions is a straightforward procedure when employing a high precision microbalance and a proper set of tools adapted for the routine analysis of several samples. Nevertheless, in order to establish a broader and more universal procedure and determine the precision of this assay, additional studies are warranted, including a more extended range of different lipids. In addition to phospholipids, the generic gravimetric assay is anticipated to be readily applicable to a wider variety of lipids such as sphingolipids, sterols, acylglycerols, and fatty acids.
6. Conclusions We here describe a detailed procedure for the gravimetric analysis of the concentrations of lipid stock solutions. The results presented here establish this assay as a fast and very accurate technique when compared to total phosphorus determination by methods such as Bartlett’s or 31 P NMR. Acknowledgments This work was supported by EU FP7 project Sonodrugs (NMP4-LA-2008-213706), Finnish Academy, and Sigrid Jusélius Foundation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemphyslip. 2012.06.005. References Banno, B., Ickenstein, L.M., Chiu, G.N.C., Bally, M.B., Thewalt, J., Brief, E., Wasan, E.K., 2010. The functional roles of poly(ethylene glycol)-lipid and lysolipid in the drug retention and release from lysolipid-containing thermosensitive liposomes in vitro and in vivo. Journal of Pharmaceutical Sciences 99, 2295–2308. Bartlett, G.R., 1959. Phosphorus assay in column chromatography. Journal of Biological Chemistry 234, 466–468. Brockman, H.L., Jones, C.M., Schwebke, C.J., Smaby, J.M., Jarvis, D.E., 1980. Application of a microcomputer-controlled film balance system to collection and analysis of data from mixed monolayers. Journal of Colloid and Interface Science 78, 502–512. Christie, W.W., 1992. Evaporative light-scattering detectors in the analysis of lipids by HPLC. Available from: http://lipidlibrary.aocs.org/topics/detect92/ index.htm. (accessed on 28.05.2012). de Smet, M., Langereis, S., van den Bosch, S., Grüll, H., 2010. Temperature-sensitive liposomes for doxorubicin delivery under MRI guidance. Journal of Controlled Release 143, 120–127. Dubinnyi, M.A., Lesovoy, D.M., Dubovskii, P.V., Chupin, V.V., Arseniev, A.S., 2006. Modeling of 31P NMR spectra of magnetically oriented phospholipid liposomes: a new analytical solution. Solid State Nuclear Magnetic Resonance 29, 305–311. Fahy, E., Subramaniam, S., Brown, H.A., Glass, C.K., Merrill Jr., A.H., Murphy, R.C., Raetz, C.R.H., Russell, D.W., Seyama, Y., Shaw, W., Shimizu, T., Spener, F., van Meer, G., VanNieuwenhze, M.S., White, S.H., Witztum, J.L., Dennis, E.A., 2005. A comprehensive classification system for lipids. Journal of Lipid Research 46, 839–861. Fenske, D.B., Cullis, P.R., 2005. Entrapment of small molecules and nucleic acid-based drugs in liposomes. Methods in Enzymology 391, 7–40. Fiske, C.H., Subbarow, Y., 1925. The colorimetric determination of phosphorus. Journal of Biological Chemistry 66, 375–400. Jendrasiak, G.L., Hasty, J.H., 1974. The hydration of phospholipids. Biochimica et Biophysica Acta 337, 79–91. Jendrasiak, G.L., Smith, R.L., Shaw, W., 1996. The water adsorption characteristics of charged phospholipids. Biochimica et Biophysica Acta 1279, 63–69. Moore, J.D., Caufield, W.V., Shaw, W.A., 2007. Quantitation and standardization of lipid internal standards for mass spectroscopy. Methods in Enzymology 432, 351–367. Padday, J.F., Pitt, A.R., Pashley, R.M., 1975. Menisci at a free liquid surface: surface tension from the maximum pull on a rod. Journal of the Chemical Society, Faraday Transactions 1 (71), 1919–1931. Sabatini, K., Mattila, J.P., Megli, F.M., Kinnunen, P.K.J., 2006. Characterization of two oxidatively modified phospholipids in mixed monolayers with DPPC. Biophysical Journal 90, 4488–4499. Smaby, J.M., Brockman, H.L., 1991. Evaluation of models for surface pressure-area behavior of liquid-expanded monolayers. Langmuir 7, 1031–1034. Smaby, J., Kulkarni, V., Momsen, M., Brown, R., 1996. The interfacial elastic packing interactions of galactosylceramides, sphingomyelins, and phosphatidylcholines. Biophysical Journal 70, 868–877. Smaby, J.M., Momsen, M.M., Brockman, H.L., Brown, R.E., 1997. Phosphatidylcholine acyl unsaturation modulates the decrease in interfacial elasticity induced by cholesterol. Biophysical Journal 73, 1492–1505. Sotirhos, N., Herslöf, B., Kenne, L., 1986. Quantitative analysis of phospholipids by 31P NMR. Journal of Lipid Research 27, 386–392. Söderlund, T., Lehtonen, J.Y.A., Sievi, J.J., Kinnunen, P.K.J., 1997. Formation of a macroscopic membrane structure by liposomes containing cationic lipids
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Contents lists available at SciVerse ScienceDirect
Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip
Gravimetric determination of phospholipid concentration Roberto Tejera-Garcia a , Lisa Connell b , Walter A. Shaw b , Paavo K.J. Kinnunen a,∗ a Helsinki Biophysics and Biomembrane Group, Department of Biomedical Engineering and Computational Science, School of Sciences, Aalto University, P.O. Box 12200, FI-00076 Espoo, Finland b Avanti Polar Lipids, Inc., Alabaster, AL 35007, United States
a r t i c l e
i n f o
Article history: Received 23 April 2012 Received in revised form 6 June 2012 Accepted 18 June 2012 Available online 5 July 2012 Keywords: Phospholipid concentration Phosphate assay Microbalance Gravimetric analysis
a b s t r a c t Accurate determination of lipid concentrations is an obligatory routine in a research laboratory engaged in studies using this class of biomaterials. For phospholipids, this is frequently accomplished using the phosphate assay (Bartlett, G.R. Phosphorus Assay in Column Chromatography. J. Biol. Chem. 234, 466–468, 1959). Given the purity of the currently commercially available synthetic and isolated natural lipids, we have observed that determination of the dry weight of lipid stock solutions provides the fastest, most accurate, and generic method to assay their concentrations. The protocol described here takes advantage of the high resolution and accuracy obtained by modern weighing technology. We assayed by this technique the concentrations of a number of phosphatidylcholine samples, with different degrees of acyl chain saturation and length, and in different organic solvents. The results were compared with those from Bartlett assay, 31 P NMR, and Langmuir compression isotherms. The data obtained show that the gravimetric assay yields lipid concentrations with a resolution similar or better than obtained by the other techniques. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Accurate determination of lipid stock solution concentrations is mandatory in both basic as well as applied studies on lipids. More specifically, the biophysical properties of lipid monolayers, supported lipid bilayers, and liposomes are in most cases profoundly influenced by even minor variations in the molar ratios of multicomponent lipid mixtures (Tejera-Garcia et al., 2011). As an example, the drug release characteristics of thermosensitive liposome formulations are critically dependent on even minor differences in the relative contents of their constituent lipids, reflecting the sensitivity of lipid membrane thermal phase behavior to the exact stoichiometry of its components (Banno et al., 2010; Wang and Thanou, 2010). Even when rigorously handled according
Abbreviations: DPPC, 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine; C, in chloroform; E, in ethanol; X, in a chloroform, methanol, water mixture (65:35:8, by volume); Di-18:2 PC, 1,2-di-(9Z,12Z-octadecadienoyl)-sn-glycero-3phosphocholine; A, total monolayer area; LC, liquid condensed phase; LE, liquid expanded phase; N, number of molecules; 31 P NMR, phosphorus-31 nuclear magnetic resonance; V, volume; , surface pressure. ∗ Corresponding author at: Helsinki Biophysics and Biomembrane Group (HBBG), Department of Biomedical Engineering and Computational Science, Aalto University, Otakaari 3J, 3rd floor, 02150 Espoo, Finland. Tel.: +358 5 05404600; fax: +358 9 470 23182. E-mail address: paavo.kinnunen@aalto.fi (P.K.J. Kinnunen). 0009-3084/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemphyslip.2012.06.005
to the lipid manipulation directives (Moore et al., 2007), the volatility of commonly used lipid solvents such as chloroform necessitates frequent routine control of lipid stock solution concentrations. The development of a universal method for the accurate determination of lipid stock solution concentrations is not straightforward due to the chemical diversity of lipid species (Fahy et al., 2005), and because of the inherent difficulties in the preparation of pure lipid standards of stable concentration. Protocols used today for the assessment of lipid stock solution concentrations are commonly based on techniques originally developed for the characterization of more complex systems. In 1925, Fiske and Subbarow designed a phosphate assay for the analysis of urine and blood samples, employing strong acids to liberate and oxidize phosphorus in the sample, producing phosphate ions that in the presence of proper reducing reagents form colored complexes amenable to quantification by absorbance spectroscopy (Fiske and Subbarow, 1925). This assay was subsequently adapted for the analysis of a variety of different samples and improved to obtain higher sensitivity. Along these lines, the method of Bartlett is at present perhaps the most widely employed in phospholipid research (Bartlett, 1959). Due to its reproducibility, requiring no expensive reagents, and being rather fast to complete (about 3 h), the Bartlett assay can be considered as a reference procedure for assaying phospholipid concentration (Söderlund et al., 1997; Fenske and Cullis, 2005). A complete description of a modern variant of this assay can be found on the Avanti Polar
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Lipids website (http://www.avantilipids.com - Determination of Total Phosphorus). Nevertheless, this assay involves some inconveniences: i. It is time and resource consuming, requiring strict cleaning protocols, usually involving acid washing, to keep the glassware used phosphate free. A continuous supply of fresh reagents is needed to obtain consistent results, especially in laboratories where it is run as a routine. The preparation of the necessary standard curves is time consuming. ii. Most of the assay must be performed using fume hoods and protective equipment, like gloves and glasses, because of the inherent risks of handling strong acids at high temperatures. The explosion hazard associated with possible complete evaporation of the sample is controlled using glass marbles covering the tubes and constantly running water flows on the fume hood faucets to increase humidity. 31 P
NMR measures the electromagnetic energy emitted during the reorientation of the spin polarization of 31 P isotopes, in the presence of an external magnetic field, after perturbation at controlled radiofrequency. Measurement of lipid stock solution concentrations by this technique has been described (http://www.avantilipids.com - Phospholipid Analysis, 31P NMR). 31 P NMR also allows the assay of phospholipid concentration without separation or other kind of sample manipulation and even quantification of individual phospholipids in multi-component mixtures (Sotirhos et al., 1986). It produces more precise and accurate data than the Bartlett assay and has been recently refined for use with liposomes (Dubinnyi et al., 2006). However, its use for routine measurement of lipid stock solutions is limited by instrument availability and due to large amounts of material required. Several other techniques have been employed for the measurement of lipid stock solution concentrations although none of them has reached the popularity of the Bartlett phosphate assay. These include inductively coupled plasma-mass spectrometry (ICP-MS) (Moore et al., 2007; de Smet et al., 2010); and separation based techniques such as evaporative light-scattering detection (ELSD), and refractometric and spectrophotometric detectors (Christie, 1992). The limitations inherent to most of the above techniques, together with the development of affordable and precise microbalances, prompted us to develop gravimetric analysis for the measurement of lipid stock solution concentrations (Zhao et al., 2005), described here in detail. Its simplicity and precision make this assay superior to the other methods. The possibility of eventually recovering the pure dry lipid after the measurement, as well as no need for specific and in some cases expensive laboratory equipment, explicit reagents or previous user expertise, are some of the inherent advantages of this method. The evaluation of the protocol described here shows that the use of gravimetric assay performed with a modern high precision microbalance, allows to obtain data comparable or better than obtained by some of the most precise techniques available today. Our assay is more accurate than the traditional methods, which are now routinely used in most laboratories.
2. Materials 1,2-Dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-di-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine (di -18:2 PC) in the indicated solvents, C: chloroform, E: in ethanol, and X: in a chloroform, methanol, water mixture (65:35:8, by volume), were from Avanti Polar Lipids (Alabaster, AL). Other chemicals were of analytical grade and from standard sources.
3. Experimental 3.1. Bartlett phosphorus assay Solutions of DPPC and di-18:2 PC in solvents C, E, and X, each at concentrations of 2.0, 0.8, and 0.4 mg/mL were prepared for analysis by the Barlett phosphorus method as described (http://www.avantilipids.com - Determination of Total Phosphorus). Approximately 0.1 mol of each sample was added to the bottom of respective reaction tubes in triplicate using a glass gastight Hamilton syringe (Hamilton Company Reno NV). Solutions were evaporated to dryness at room temperature with a gentle flow of grade 5 nitrogen gas. Standards of 0.0325–0.2280 mol were prepared in like manner by delivering appropriate volumes of 0.65 mM stock solution (Sigma, St. Louis, MO). After reaction, the solutions were assayed on an Agilent (Englewood, CA) model 8453 diode array spectrophotometer scanning from 190–850 nm. Phosphorus concentrations were calculated by reading the absorbance intensity at 820 nm plotted from the linear standard curve and converted to mg/mL and mM concentrations using the molecular weights of 734.04 g/mol for DPPC and 782.08 g/mol for di-18:2 PC. Precision for triplicate assay was <2%. 3.2.
31 P-nuclear
magnetic resonance
The DPPC and di-18:2 PC solutions described above were assayed by 31 P NMR after transferring 0.5 mL (0.25–1.5 mol) to 1.0 mL volumetric tubes with a glass gas tight syringe. These solutions were dried to a residue using a gentle stream of grade 5 nitrogen at room temperature. The residues were dissolved in 1 mL of sodium cholate detergent. Solutions were assayed on a Bruker AvanceTM 400 MHz NMR (Bruker Daltonik, Bremen, Germany) system calibrated with an external standard of dioleoyl phosphatidylcholine (18:1 PC) (Avanti Polar Lipids, Alabaster AL). Quality control samples of dioleoyl phosphatidylethanolamine assayed with 100 ± 2% accuracy. 3.3. Gravimetric assay In this study the microbalance and the set of accessories employed in the experiments described were contained in the SuperG lipid gravimetric analysis kit (Kibron Inc., Helsinki, Finland). In addition to the microbalance, the kit includes a set of aluminum sample pans for weighing (max. capacity, 80 l), a pair of inverse clamp curved tweezers for pan handling, avoiding contact with the internal surface of the sample pans, and a storage box with labeled separate compartments for sample identification. The assay is performed as a series of default steps following the instructions of the microbalance’s software: 1. The needed number of pans was selected and placed each into the assigned numbered compartments according to the number of samples to be measured. 2. The tare masses of the empty pans were measured and recorded in a log file of the software supervising the assay. After each measurement the pans were returned to their respective compartment in the box. 3. Next, boxes containing the empty pans were placed under a moderate stream of nitrogen and a specified volume (from 10 to 80 l) of lipid sample was deposited drop by drop in each pan using a microsyringe (Hamilton, Bonaduz, Switzerland). Subsequently, the samples were maintained under nitrogen flow until complete evaporation of the liquid phase, taking approximately 5–30 min, depending on the lipid type, solution volume, and nitrogen flow rate. As an alternative, and in order to prevent oxidation especially when working with polyunsaturated
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lipids, this procedure was also performed inside a nitrogen saturated atmosphere without the need of a continuous nitrogen flow in the process (see Recording Langmuir-film compression isotherms below). 4. Once the evaporation was complete, the samples were placed under reduced pressure (∼4 kPa) to remove possible traces of solvent for approximately 2 h. Comparison of the mass measured at different times allows to estimate the optimum time at reduced pressure for removal of solvent traces. 5. Finally, the masses of the different pans containing the dry lipid residues were measured by the microbalance, with the dry lipid net mass obtained after subtraction of the respective empty pan tare weight. The software then calculated the masses and molar concentrations of the lipids, according to the volumes and molecular weights provided by the user and stored in the database of the software. The weight of the hydration water on the lipid molecules headgroups was automatically subtracted from the lipid mass in all the experiments. The subtraction of this mass is important especially when comparing concentrations measured gravimetrically with those obtained by techniques measuring the amount of anhydrous phosphorus, such as NMR or phosphate assay. The precise percentage (weight) of water of hydration present in a lipid sample is usually obtained from the lipid manufacturer. The specific value of the water of hydration for the phosphatidylcholines employed here was specified as 4.4% of the total weight, which corresponds to approximately two molecules of water per head group. In cases where precise value is not available, approximate percentage of the water of hydration can be found in literature (Jendrasiak and Hasty, 1974; Jendrasiak et al., 1996). The error introduced is usually of the order of ±1 water molecule which can decrease the precision of the concentrations determined gravimetrically. In the present study this decrease in precision would represent an extra 1% in the absolute error associated to the concentrations determined 3.4. Recording Langmuir-film compression isotherms As an independent technique we additionally assessed the concentrations of the samples from compression isotherms (surface pressure, , vs. monolayer area, A) of lipid monolayers recorded at a gas/water interface. The measurement of was performed as ( 0 – ), 0 and being the interfacial tensions in the absence and in the presence of the lipid monolayer, respectively. In this study all compression isotherms ( vs. A) were recorded using a Langmuir trough (MTXL, Kibron Inc., Helsinki, Finland) equipped with a microbalance and Wilhelmy-Padday rod probe (Padday et al., 1975). The aqueous subphase solution was 40 ml of 10 mM KH2 PO4 , 100 mM NaCl in deionized filtered water (Direct-Q UV; Millipore, Jaffrey, NH), yielding a total surface area of 228 cm2 . This buffer makes the phospholipid hydration shells uniform (Sabatini et al., 2006) and was prepared daily prior every set of experiments and kept at 4 ◦ C. All measurements were performed at room temperature (24 ◦ C), inside a controlled atmosphere chamber (850 Series, Plas-Labs Inc., Lansing, MI, USA) saturated with nitrogen to avoid oxidation when working with polyunsaturated phospholipids. Saturation was achieved by keeping a constant nitrogen flow rate, while discharging the chamber at the same flow using the chamber’s vacuum pump. Complete saturation was considered after recording levels of oxygen below 2% with a dissolved oxygen meter (ISO2 ; WPI, Stevenage, UK). The chamber’s taps were closed and the experiments were performed with continuous monitoring of the stability of the oxygen levels. In this nitrogen atmosphere, a microsyringe (Hamilton, Bonaduz, Switzerland) was filled with the indicated amount of the stock solution and the main stock was hermetically closed
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to diminish possible evaporation. At this point, part of the volume of original solution in the microsyringe was separated and stored for later assay by gravimetric analysis. The sample in the microsyringe was carefully deposited onto the gas/water interface in the trough, whereafter the monolayer was allowed to stabilize for 10 min for complete evaporation of the solvents. Subsequently, two symmetrically moving Teflon barriers were used to compress the lipid monolayer at a rate of 3.6 cm2 /min, allowing reorientation and relaxation of the lipids during compression. The amount of lipid solution deposited for each sample was estimated by an initial coarse measurement of dry lipid content after evaporation in air over a hot plate (50 ◦ C). All experiments were performed twice using two aliquots of the same sample. If differences above 1 mN/m, 2 A˚ 2 /molecule, or 20 mV were found a third isotherm was recorded. For DPPC, the values of A corresponding to the cross-sectional limiting area, obtained from the extrapolation to zero surface pressure of the linear part of the compression isotherm at surface pressures around 30 mN/m, were employed for the calculation of the number of molecules (N) in the monolayer, after dividing this value by the respective areas per molecule obtained from data in the literature (50 A˚ 2 per DPPC molecule when extrapolating from data at surface pressures around 30 mN/m – see Fig. S1, panel A, top) (Smaby et al., 1996). The number of molecules derived was then employed to calculate the concentration of the original solution by dividing N by the volume (V) of the liquid solution applied onto the interface. For validation of the lipid concentration obtained, the area per molecule corresponding to the onset of the liquid expanded (LE) to liquid condensed (LC) phase transition was obtained from the recorded compression isotherms and compared with the corresponding data in the literature (71 A˚ 2 per DPPC molecule – see Fig. S1, panel A). These areas were identified as changes from positive to negative in the values of the third derivative of the vs. A function, as described by Brockman et al. (1980). For di-18:2 PC, the lipid concentrations were obtained as N/V. However, di-18:2 PC does not form LC or solid phases under the experimental conditions used. Therefore, it was not possible to use the cross-sectional limiting area or critical points of the isotherms for the calculation of N. Instead, N was obtained by fitting an adjustable parameter in the following monolayer equation of state for the LE region (Wolfe and Brockman, 1988): =
qkT ln ω1
1 f
1+
ω1 A/N − ω0
(1)
where k is the Boltzmann constant, T is temperature, ω0 the area of dehydrated lipid ( = ∞), ω1 = 9.65 A˚ 2 is the surface area occupied by one water molecule at the interface, and q and f are the activity coefficients of lipid and water, respectively. The values for di-18:2 PC of ω0 , f, and q, obtained from data in the literature (Smaby et al., 1997), were 44.86, 1.13, and 3.46 A˚ 2 , respectively. This value of ω0 was introduced in Eq. (1) and the values of f and q, obtained as fitting parameters for the isotherms recorded, and compared with the values in the literature for validation. Approximate correlation between q and f (ln q > 7.1 − 5.2f) has been proven for pure lipids (Smaby and Brockman, 1991). We consider this correlation as additional support on the accuracy of the obtained values of N. 4. Results 4.1. Characteristics of the SuperG microbalance The SuperG microbalance is routinely calibrated using an OIML class E1 standard weight of 1 mg (KERN & Sohn GmbH, Balingen, Germany) prior to its daily use, following the instructions provided by the instrument’s software. The precision (repeatability) of the
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Fig. 1. Standard deviations for ten readings of up to six standard 1 mg weights. Linearity of the balance is depicted in the inset.
instrument is provided by the manufacturer and is given according to respective sensitivity stability values. However, these values are approximate only and in order to exclude possible malfunction and to have confidence on the accuracy and precision of the dry weights measured, it is recommended to evaluate these parameters when working with an instrument in intensive use. The precision of the microbalance was evaluated as the standard deviation for ten separate measurements of a reference pan loaded with up to six 1 mg standard weights. Below 2 mg, the microbalance’s precision is about ±0.2 g (Fig. 1). Above this mass the precision decreases with the increase in load going from ±1.5 g for samples of around 3 mg up to ±2.5 g for samples of around 6 mg. The accuracy of the microbalance measurements is guaranteed as 100%, within the error interval given by the instrument’s precision, by the use of certified measurement standards. For the gravimetric assay the masses of the standard aluminum pans were first recorded. The same pans were then used to apply known volumes of the lipid stock solutions and for recording the dry weight of the lipids after evaporation of the solvents. In this way the net mass of dry lipid was obtained for each sample. 4.2. Bartlett’s assay and 31 P NMR Comparison of the lipid concentrations measured for the different solutions using the Bartlett and 31 P NMR methods reveals minor differences between the concentrations measured by these methods (Fig. 4). For most measurements 31 P NMR yields a higher precision with smaller error than the Bartlett assay.
5346 ± 6 mm2 for DPPC monolayers spread from C: chloroform, E: ethanol, and X: chloroform, methanol, water mixture (65:35:8, by volume), respectively. Using a limiting cross-sectional area of DPPC of 50 ± 1 A˚ 2 per molecule, these measured areas correspond to 18. 7 ± 0.4, 19.2 ± 0.4, and 17.8 ± 0.4 nmoles of DPPC, for the monolayers spread from the above solvents [19]. The original molar lipid concentration on each solution could therefore be determined (see Supplementary Material) as the ratio between the calculated monolayer lipid content and the corresponding volume of solution employed for monolayer deposition, yielding 2.67 ± 0.09, 0.51 ± 0.02, and 1.11 ± 0.02 mM for DPPC in C, E, and X, respectively (Fig. 4). As a further control for the calculated amounts of DPPC in the Langmuir films, the area per molecule corresponding to the onset of the LE/LC phase transition was obtained as described above, corresponding to monolayer areas of 8033 ± 6, 7967 ± 6, and 7477 ± 6 mm2 for films spread from C, E, and X, respectively (Fig. 2, top row, blue dashed line). For the DPPC concentrations calculated above, these areas correspond to 71.3 ± 1.6, 68.9 ± 1.5, and 69.8 ± 1.6 A˚ 2 per molecule, which all correspond within error to the data in the literature, 71 ± 1 A˚ 2 per molecule [19], thus confirming the accuracy of the measured lipid concentrations. Compression isotherms recorded for the di-18:2 PC monolayers exhibit the gas and LE phases at the experimental conditions used (Fig. 3). By introducing into Eq. (1) the value of ω0 = 44.86 A˚ 2 calculated from the data in the literature [22] and fitting the recorded isotherms, we obtained the corresponding values for f, q, and N. The number of molecules obtained were N = 154 × 1014 , 118 × 1014 , and 180 × 1014 for the di-18:2 PC monolayers spread from C, E, and X, respectively. Taking into account the sample volumes applied onto the gas/water interface in each case, the molar lipid concentrations could be determined as the ratio between the calculated monolayer lipid contents and these volumes of applied solution, yielding 2.56 ± 0.03, 0.98 ± 0.01, and 0.5 ± 0.01 mM for di-18:2 PC in C, E, and X, respectively (Fig. 4). The values for the activity coefficients of lipid and water obtained after fitting were f = 1.13, 1.12, and 1.13; and q = 2.97, 3.68, and 3.33 for di-18:2 PC in C, E, and X, respectively. These values are in reasonable agreement with those obtained from data in the literature [22]: f = 1.13 and q = 3.46, thus confirming the accuracy of the concentrations obtained. This agreement is clearly better for the f values than for q, maybe due to the higher exposure of the water interfacial activity to minor environmental perturbations than the one of the lipid. Nevertheless, the theoretical correlation for pure systems between these two parameters (Ln q > 7.1 − 5.2f) holds quite accurately with the solutions studied, with relative errors below 15% between the values of q obtained by this relation and the ones obtained directly as fitting parameters, thus validating the accuracy of the di-18:2 PC concentrations obtained by this method.
4.3. Langmuir-film compression isotherms An independent check on the phospholipid concentrations was obtained from the analysis of the compression isotherms of lipid monolayers prepared from the indicated lipid solutions. The determination of the lipid concentrations from the recorded compression isotherms was calculated as the number of lipid molecules per monolayer divided by V, as described above. Compression isotherms recorded for DPPC monolayers prepared from different solvents exhibit the typical distinct phases appearing during compression (Fig. 2), viz.: gaseous, liquid expanded, and liquid condensed. The plateau, corresponding to the LE/LC two-phase region appears at surface pressures of ∼9 mN/m as described in the literature [19]. The values of A corresponding to the DPPC cross-sectional limiting area are indicated by the red dotted lines in Fig. 2. These areas are 5621 ± 6, 5788 ± 6, and
4.4. Gravimetric assay The lipid dry weight obtained after the deposition of 80 ± 1 l of each solution and the subsequent removal of the solvent as described above are summarized in Table 1. After dividing the
Table 1 Mass of dry lipid after solvent removal from the solutions DPPC in C, DPPC in E, DPPC in X, di-18:2 PC in C, di-18:2 PC in E, and di-18:2 PC in X.
DPPC (g) di-18:2 PC (g)
C
E
X
2.07 ± 0.08 0.41 ± 0.01
1.84 ± 0.02 0.34 ± 0.03
1.94 ± 0.03 0.37 ± 0.01
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Fig. 2. Langmuir compression isotherms, and their consecutive derivatives, for DPPC monolayers spread from (left column) chloroform, (middle) ethanol, and (right) chloroform, methanol, water mixture (65:35:8, by volume). The red dotted line indicates the limiting area for the DPPC molecule and the blue dashed line specifies the areas for the onset of the LE/LC phase transition. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
ratio between these masses and the original volume of solution by the correspondent molecular weights the respective molar lipid concentrations were as compiled in Table S1 in Supplementary material (see also Fig. 4). These concentrations were within the
concentration limits of the gravimetric assay. The maximum and minimum concentration limits as well as the errors in the concentrations determined by gravimetric assay depend on the precision of the microbalance and the microsyringe resolution, the lipid in
Fig. 3. Langmuir compression isotherms for di-18:2 PC monolayers spread from (dotted line, middle) chloroform, (solid line, left) ethanol, and (dashed line, right) chloroform, methanol, water mixture (65:35:8, by volume).
Fig. 4. Concentrations of the DPPC and di-18:2 PC solutions in C: chloroform, E: ethanol, and X: in chloroform, methanol, water (65:35:8 by volume), obtained from Bartlett’s phosphate assay, 31 P NMR, Langmuir compression isotherms and gravimetric assay.
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question and its concentration, and the volume of the sample analyzed. The computation of these limits has been implemented in the software of the microbalance used in this study, specifically designed for gravimetric analysis. The minimum concentration accessible to gravimetric analysis is limited by the minimum weight measurable by the microbalance. For SuperG this minimum weight is 1 g, which when applied in the maximum volume of sample applicable (80 l) corresponds to 1.36 and 1.28 nM for DPPC and di-18:2 PC, respectively. Depending on the assay parameters, maximum concentration limit is determined by the precision of the volumes deposited (± 0.5 l for the microsyringe employed here), which causes the accuracy of the assay to decrease at higher lipid concentrations. Results from the concentrations determined using the Bartlett assay and monolayer studies present absolute errors of up to 0.1 mM (Fig. 4 and Table S1). For DPPC and di-18:2 PC the maximum concentration limit would correspond to approximately 15.5 mM.
5. Discussion Comparison of the lipid concentrations of the solutions in different solvents measured by the methods used in this study reveal similar concentration values for each solution, in most cases with overlapping error intervals. The set of stock solutions studied shows no systematic deviation for any of the techniques in the concentration values obtained and only minor differences are evident between the different techniques in some singular cases. For DPPC solutions the concentrations obtained by the Bartlett method are slightly higher than the ones from other techniques, while the ones from 31 P NMR are lower. This is likely to be due to evaporation of the solvents which is an inherent problem when working with organic solvent solutions. In this particular case, evaporation as a source of extra error could be due to the fact that, even when same original solutions were employed for both measurements, technical limitations required the use of different ampoules for the Bartlett and 31 P NMR assays. Concentrations obtained from compression isotherms and gravimetric assay were studied simultaneously and from the same ampoule, and do show closely parallel values thus strengthening the above line of reasoning. Concentrations obtained by the gravimetric assay represent values intermediate to those measured by the other methods, yet with similar or smaller errors. 31 P NMR and Langmuir compression isotherms were both precise for the determination of lipid stock solution concentrations. However, as mentioned above, from a practical point of view the application of these techniques for routine evaluation of lipid stocks in a laboratory is not feasible. The relevance of the Bartlett method and other methods based on the determination of phosphorus contents in lipid extraction and separation processes is practical. However, for routine measurement of lipid stock solution concentrations, the time and resources that this technique require makes the gravimetric assay attractive. The microbalance employed in this study fulfilled perfectly the sensitivity requirements of the gravimetric assay. We consider this assay to represent an optimum routine tool in any laboratory for the evaluation of lipid stock solution concentrations. Accurate gravimetric determination of phospholipid concentrations in stock solutions is a straightforward procedure when employing a high precision microbalance and a proper set of tools adapted for the routine analysis of several samples. Nevertheless, in order to establish a broader and more universal procedure and determine the precision of this assay, additional studies are warranted, including a more extended range of different lipids. In addition to phospholipids, the generic gravimetric assay is anticipated to be readily applicable to a wider variety of lipids such as sphingolipids, sterols, acylglycerols, and fatty acids.
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