Quantitative determination of lipid, and of co-existing two compounds

Abstract: Lipid is determined quantitatively by treating a sample with a surfactant in an aqueous medium to obtain a liquid sample dispersion, then reacting the dispersion with a reagent containing cupric ion and bicinchoninic acid, and measuring a colored state developed by complex formation of bicinchoninic acid with cuprous ion formed from cupric ion in the presence of lipid. This method can be effectively applied to determination of amphiphilic lipid having a tendency of gathering in an aqueous medium. Further, two different species interfering with each other in quantitative determination thereof are determined respectively by conducting two different determination methods on the same sample followed by calculation using the values obtained.

Description: BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a method of quantitative determination of lipid. Related Background Art Lipids are construction components of cells or organisms. Among the lipids; phospholipid is a principal lipid in construction of various membranes in the cells , such as plasma membrane, nuclear membrane, endoplasmic reticulum membrane, mitochondrial membrane, Golgi body membrane, lysosome membrane, and the like. The phospholipid is an amphiphilic molecule having both a polar group and a hydrophobic group in the molecule. When phospholipid is suspended in water, the polar groups come to be hydrated with water molecules, and the hydrophobic groups are excluded from the aqueous environment to cause gathering of the hydrophobic groups of the lipids. Consequently, micelles, lipid bilayers having a bimolecular membrane structure, or hexagonal II structures are formed: the state of gathering depending on the balance of the volumes of the hydrated hydrophilic groups and the hydrophobic groups. Among such structures, the lipid bilayer is the basic structure of biomembranes. The bilayer of phospholipid forms a closed vesicle (liposome), which is capable of incorporating membrane protein or the like as a constituent thereof and enclosing an aqueous phase therein. Therefore, the phospholipid bilayer structure is frequently employed as a model of a biomembrane for substance permeation, information transmission, and so forth. Further, the liposome is promising for use for a medicine capsule since the liposome is capable of retaining a water-soluble substance in its internal aqueous phase. Many methods are known for quantitative determination of phospholipid, including a wet combustion method (Hoeflmayr, J. and Fried, R; Med. und Ern., 7 , 9-10 (1966)) and a choline oxidase-phenol method (Takayama, M., Itoh,S., Nagasaki, T. and Tanimizu, I., Clin. Chim. Acta 79 93-98 (1977)). In the wet combustion method, the phospholipid is determined by adding sulfuric acid and a permanganate salt to a sample; heating the mixture in a boiling water bath to liberate the constituting phosphoric acid; adding ammonium molybdate and a reducing agent thereto; and measuring light absorbance caused by molybdenum blue formed thereby. In the choline oxidase-phenol method, the phospholipid is determined by reacting phospholipase D with the phospholipid in a sample to liberate the constituting choline; further reacting choline oxidase with the resulting choline to form betaine and hydrogen peroxide; and measuring light absorbance of the product of a quantitative condensation oxidation of phenol with 4-amino-antipyrine caused by hydrogen peroxide in the presence of peroxidase. The above-mentioned wet combustion method has disadvantages of troblesomeness in the operation of sequential addition of several reagents, and danger of bumping and measurement error by evaporation owing to heating in the presence of sulfuric acid in a boiling water bath. The choline oxidase-phenol method has disadvantages of instability of the enzyme, need for storing the reagents in a cold and dark condition, and insufficiency of reproducibility of measurement data obtained during lapse of time. Generally, absorption spectrochemical quantitative analysis, in which the absorbance is measured at an absorption wavelength characteristic of the object of analysis in the sample, is widely used practically in quantitative determination of a substance because of simplicity of operation of direct measurement of absorbance, and needlessness of reacting the analysis object with another substance that enables the use of the sample later for another use. However, this method of analysis has not been employed in the determination of lipids, because lipids such as phospholipid constituted of amphiphilic molecules form in an aqueous medium an aggregate in various state which scatters light and prevents precise assay of lipid concentration by light absorbance. The constitution components of living matter such as lipids, proteins, nucleic acids, sugars are frequently handled in a coexisting state. Therefore, quantitative determination of such coexisting substances is an important operation. Biomembranes have in a structure of a bilayered membrane mainly constituted of lipid molecules, and protein gathers therein by non-covalent bonds, thereby functioning as a barrier for maintaining the interior medium. The ratio of lipid to protein in biomembranes differs greatly depending on the respective membranes. In mitochondrial membranes, for example, protein is contained 4 times as much as lipid. On the contrary, in some biomembranes, lipid is contained several times as much as protein. Accordingly, the ratio of lipid to protein in biomembrane in respective organs is an important object of study. Various biomembrane models in which lipid and protein are coexisting are widely used in the study of the structure and the function of the biomembranes. The examples include proteoliposome which has closed vesicles constituted of artificial bimolecular lipid membrane containing therein protein; fine particles of glass beads or a high polymer which have lipid or protein adsorbed on the surface thereof; a planar lipid membrane formed in a small hole in a substrate made of teflon or the like (a black lipid membrane), or a planar bimolecular film reconstructed by sticking or patch-pipeting into which protein is incorporated; and Langmuir-Blodgett built-up films (LB built-up films) in which a lipid-protein coexisting system is built up in layers in a molecular level. In these artificial films also, the determination of the lipid and the protein is important in evaluating the function thereof. If the optimum ratio of lipid to protein in natural, or artificially synthesized membrane structures can be obtained by quantitative determination, and a membrane can be synthesized effectively according to the derived ratio, then various membrane structure can be industrially synthesized at low cost without wasting valuable protein. Further examples of lipid-protein coexisting systems include liposome, i.e., a closed vesicle composed of phospholipid membrane, in which a functional protein such as water-soluble enzyme is enclosed; and emulsions in which lipid and protein are simply mixed in a liquid medium. Heretofore, in the determination of lipid and protein in a coexisting system, the determination of lipid is affected by the presence of protein, and the determination of protein is affected by the presence of lipid. Therefore, lipid and protein could not be precisely determined in a coexisting state. In determination of lipid by the above-mentioned absorbance measurement, the influence of the absorbance of coexisting protein is not negligible. Various methods for determining protein are known, including colorimetry utilizing a metal ion, UV absorption method based on tyrosine and triptophan, and UV absorption method based on peptide bonds. A typical method of the colorimetry is a Lowery method (J. Biol. Chim., vol. 193, p265 (1951)) employing a phenol reagent. This method utilizes a blue color developed by reaction of a phenol with protein. The main component of the reagent is phosphomolybdate or phosphotungstate, a complicated complex formed from the metal oxide and phosphoric acid. This complex reacts with a reducing agent such as protein to develop blue color of phosphomolybdate blue or phosphotungstate blue. The protein is determined by measurement of the absorbance caused by the color. In a color reaction method called a BCA method, protein present converts a cupric ion into a cuprous ion in an alkaline medium, and the cuprous ion forms a purple complex compound with bicinchoninic acid molecule (i.e., BCA: 4,4'-dicarboxy-2,2'-biquinoline). The protein is determined according to the absorbance of this complex compound. On the contrary, the UV absorption method for determining protein includes measurement of absorption at 280 nm caused by tyrosine or triptophan, and measurement of absorption at 215 to 225 nm caused by the peptide linkage. The both measurement methods are advantageous in that the operation is simple and the sample is not lost in comparison with color development methods, but are disadvantageous in that the measurement of protein is based on the content of tyrosine or triptophan and the determination result varies greatly depending on the kinds of the protein and is susceptible to an interfering substance such as nucleic acid which has absorption in this absorption region. In these known methods of determining protein, coexisting lipid also undergoes reaction to develop color. For example, in the Lowry method and other methods employing a phenol reagent, the phosphomolybdate is reduced by the protein to develop a blue color of phosphomolybdate blue, and simultaneously the coexisting lipid which exhibits reduction activity like the protein develops color similarly. Accordingly, in the quantitative determination of each of lipid and protein coexisting in a system, they have to be separated before the determination of lipid and protein. The separation is usually conducted by an extraction method in which the lipid component in the sample is dissolved in an organic solvent layer and is recovered as an organic solvent fraction, and the protein component insoluble in the organic solvent is recovered as an aqueous fraction. The obtained organic solvent fraction is concentrated and evaporated to dryness, and is quantitatively determined as the lipid fraction. The aqueous fraction containing the protein is concentrated and the protein therein is quantitatively determined by various methods. Thus the conventional determination of lipid and protein coexisting in a system is extremely complicated. In conventional methods, in spite of the complicated operations, the protein is liable to enter the organic solvent layer, and the lipid is liable to enter the aqueous layer in some proportions since the extraction with an organic solvent is based on equilibrated partition, which causes a determination error frequently. Moreover, loss of the sample is not negligible in the extraction process, so that a relatively large amount of a sample is required. Furthermore, in determination of lipid and protein contained in a membrane structure, strongly hydrophobic protein such as membrane protein may contaminate the organic solvent fraction in high probability, and the lipid and the protein may possibly exist in an unseparated state in the organic layer, which prevents precise quantitative determination. The above discussion relates to a two component system containing lipid and protein. The same discussion may be made on a two-component system containing two components selected from lipids, sugars, nucleic acids, pigments and various medicines. US-A-4,839, 294 discloses an enzyme free, non-hydrolyzing assay useful for determination of lipid content of a lipid-containing fluid which comprises forming a water insoluble complex of a charged dye and an oppositely charged detergent, incubating the dye-detergent complex in an aqueous dispersion of a lipid-containing fluid thereby imparting a characterictic color to the lipid containing fluid, said characteristic color exhibiting light absorbance, and measuring the light-absorbance of the characteristic color whereby the lipid content of the fluid is determined. SUMMARY OF THE INVENTION An object of the present invention is to provide a highly simplified method for determining, in an aqueous medium, lipid, particularly a simplified method for determining amphiphilic lipid which gathers in the aqueous conditions. The objects above are achieved by providing a method according to claim 1. A preferred embodiment is set forth in claim 2. According to the present invention, there is provided a method for quantitative determination of lipid, comprising preparing a liquid sample dispersion by treating a sample with a surfactant in an aqueous medium, reacting the liquid sample dispersion with a reagent containing cupric ion and bicinchoninic acid to reduce the cupric ion to cuprous ion, and measuring a colored state developed by complex formation of bicinchoninic acid with cuprous ion. DESCRIPTION OF THE PREFERRED EMBODIMENT Firstly, the method of determining lipid in the present invention is described below. In any of the lipid determination methods in the present invention, the lipid is treated with a surfactant to form a micelle composed of the lipid and the surfactant to enable the quantitative determination. For example, in the case of an amphiphilic lipid such as phospholipid, lipid aggregate formed in an aqueous medium is treated with a surfactant to form a micelle state composed of the lipid and the surfactant. The suitable treatment with a surfactant is a supersonic treatment or a water-bath heating treatment. One minute of supersonic treatment, or heating on a water bath at 60°C for 10 minutes or longer is sufficient. An unknown sample and a standard sample should be treated in the same manner. By such a treatment, the phospholipid with a surfactant is brought into a micelle state in correspondence with the lipid concentration regardless of the form of the aggregate such as liposome, a planar membrane, and a hexagonal II structure. In the method of quantitative determination of lipid, the lipid with a surfactant in a micelle state functions as a reducing agent to convert cupric ion into cuprous ion in an aqueous medium. The resulting cuprous ion forms with sodium salt of bicinchoninic acid (4,4'-dicarboxy-2,2'-biquinoline) a complex compound in purple color. Since this reaction proceeds quantitatively, lipid can be determined quantitatively by measurement of absorbance caused by the color of the complex compound. The surfactant useful in this determination includes nonionic surfactants, and anionic surfactants, but are not particularly limited. The specific examples are Triton X-100, SDS (sodium dodecylsulfate), Briji 35, octylglucoside (n-octyl-β-D-glucopyranoside), CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate), HECAMEG (6-O-(N-heptylcarbamoyl)-methyl-α-D-glucopyranoside), cholic acid, deoxycholic acid, and the like. The color reaction of cupric ion with bicinchoninic acid is known also as a method of determination of protein by utilizing the reducing action of protein (Anal. Biochem., 150, p76 (1985)). This method is based on the principle that the cupric ion transforms into cuprous ion in the presence of protein in an alkaline medium, and the resulting cuprous ion forms a purple complex compound with the bicinchoninic acid molecule. The inventors of the present invention found that this reaction is sensitive not only to protein but also to lipid exhibiting similar reactivity, and completed the present invention on this finding. Not all methods of quantitative determination of protein are applicable to quantitative determination of lipid. The present invention is characterized by the finding that the method for determining protein by use of bicinchoninic acid is applicable to quantitative determination of lipid. For example, the aforementioned Lowry method of protein determination, which utilizes reducing action of protein, may be expected to be useful for lipid determination by use of lipid exhibiting reducing action in place of protein. Actually, however, the sensitivity to lipid is as low as about 1/100 times that to protein. On the contrary, the sensitivity of the method of the present invention is only slightly lower than that to protein, about 1/3 times that to protein, being more sensitive than conventional quantitative lipid determination. Further this method is simple in operation. Therefore, the second method of determination of lipid of the present invention is highly practical when considered as a whole. In the method of the present invention, a liquid sample dispersion formed by treatment with a surfactant is reacted with a color reagent containing bicinchoninic acid and cupric ion, and the absorbance caused by the purple color developed by the presence of lipid is measured. The bicinchoninic acid and cupric ion are added in excess of the amount for reaction of the entire lipid in the sample. Usually, the final concentration of the bicinchoninic acid is about 1 %, and that of cupric ion is from about 0.01 to 0.1 %, and the sample dispersion is diluted so as to be measured in this reagent concentration. The reaction condition may be suitably selected. For example, heating treatment may be practiced at about 60°C for about one hour, and cooled to room temperature, and the absorbance may be measured at a wavelength of, e.g., 526 nm. This method for determination of lipid has advantages that the reagents are added all at once and the colored substance is stable for a long time, the sensitivity is higher than in other lipid determination methods, and the operation is very simple. In the above method of lipid determination of the present invention, the lipid is treated with a surfactant in an aqueous medium. However, the lipid to be determined is not required to be originally in a state of suspension in an aqueous medium. For example, if the sample is solid, the determination is made feasible by treating the solid sample with a prescribed volume of an aqueous surfactant solution. The composition of the aqueous medium for constituting the reaction system is not particularly limited, if the ionic strength and the pH are within the ranges in which the solubilizing ability of the surfactant is maintained. The unknown sample and the standard sample should be treated at the same pH and the same ionic strength or salt concentration. In the method of determination of the present invention, the optimum pH value for color reaction is 11.25. Therefore, it is undesirable to use a strongly acidic solution which will shift greatly the pH of the medium to an acidic side. The present invention is described in more detail by reference to Examples. Reference Example 1 (outside the scope of the present invention) In an eggplant-shape flask, 0.5 mℓ of a solution of 30 mg/mℓ of asolectin (soybean phosphatidylcholine, type IV-S; made by Sigma Co.) in chloroform was placed. The solvent was evaporated off by use of a rotary evaporator, and then completely removed completely in a desiccator in vacuum to form a thin film of lipid. Thereto, 1.0 mℓ of aqueous 10mM potassium chloride solution was added and treated with a vortex mixer for 5 minutes to disperse the thin lipid film. Then it was treated with a water-bath type ultrasonic oscillator (Sonifier Type B-15, using cup horn, made by Branson Co.) for 30 minutes to obtain a liposome dispersion (15 mg/mℓ). The liquid liposome dispersion thus prepared was found to have a particle size distribution in the range of from 30 to 260 nm (average particle diameter of 190 nm) by use of a dynamic light-scattering particle size analyzer. (DLS-700, Otsuka electronics Co. LTD.) This liposome dispersion was diluted with aqueous 10 mM potassium chloride solution containing 1 by weight of octylglucoside (n-octyl-β-D-glucoside; made by Wako Pure Chemical Industries, Ltd.) to various concentrations and the diluted dispersion was treated with the water-bath type supersonic oscillator for one minute to obtain phospholipid standards in a concentration range of from 0 to 1.5 mg/mℓ. The standard phospholipid liquid was put into a quartz cell having ah optical path of 1 cm, and the absorbance was measured at 240 nm by means of a spectrophotometer (UV-VIS-NIR Recording Spectrophotometer UV 3100S, Shimadzu Corporation). The absorbance values were plotted as a function of the phospholipid concentration by use, as the reference, of the absorbance of aqueous 10mM potassium chloride solution containing 1% octylglucoside. Thus a straight line passing the origin was obtained. From the gradient of the line, the following relation was valid between the phopholipid concentration and the absorbance: (Absorbance) = 0.400 × (phospholipid concentration, mg/mℓ) Accordingly, the quantity of phospholipid in an unknown sample can be determined by treating the unknown sample in the same manner as above, measuring the absorbance, and calculating with the above equation. Reference Example 2 (outside the scope of the present invention) A liquid liposome dispersion was prepared in the same manner as in Reference Example 1. The dispersion was frozen by immersion in liquid nitrogen, was thawed by leaving standing at room temperature, then was subjected to supersonic treatment for one minute. This freezing and thawing operation was conducted six times, whereby a liposome liquid dispersion having relatively large particle diameter could be prepared. The particle diameter distribution was found to be in the range of from 130 to 520 nm (average particle diameter: 280 nm) by measurement in the same manner as in Example 1 by means of a dynamic light-scattering particle size analyzer. The liposome dispersion thus prepared was diluted with an aqueous 10 mM potassium chloride solution containing 1 % by weight of octylglucoside in the same manner as in Reference Example 1, the diluted dispersion was treated with the water-bath type supersonic oscillator for one minute in the same manner as in Example 1 to obtain phospholipid standards in a concentration range of from 0 to 1.5 mg/mℓ. The absorbance of the standard phospholipid was measured at 240 nm. The absorbance values were plotted as a function of the phospholipid concentration by use, as the reference, of the absorbance of aqueous 10mM potassium chloride solution containing 1% octylglucoside. Thus a straight line passing the origin was obtained. From the gradient of the line, the following relation was valid between the phopholipid concentration and the absorbance: (Absorbance) = 0.400 × (phospholipid concentration, mg/mℓ) Accordingly, the quantity of phospholipid in an unknown sample can be determined by treating the unknown sample in the same manner as above, measuring the absorbance, and calculating with the above equation. Example 3 Phospholipid standard liquid was prepared in the concentration range of from 0 to 50 µg/mℓ in the same manner as in Reference Example 1. To each 0.5 mℓ of the phospholipid standard solutions, an equal amount of a BCA color developing solution (Micro BCA Protein Assay Reagent Kit purchased from Pierce) was added and mixed, and the heated at 60°C for one hour. The mixture was cooled to room temperature. Absorbance was measured at 562 nm with a cell having optical path of 1 cm. From the measured absorbance, the absorbance of the control containing no liposome was subtracted. The resulting absorbance was plotted taking the absorbance on the ordinate and the liposome concentration on the abscissa. The plots are on a line passing the origin, the gradient of the line being 3.89 × 10 -2 . Accordingly, the quantity of lipid (X µg/mℓ) in an unknown sample can be determined by treating the unknown sample in the same manner as above, measuring the absorbance (Y), and calculating with the relation: Y = 3.89 × 10 -2 X. The present invention greatly simplifies determination of phospholipid which takes much labor conventionally. In particular the present invention provides an extremely simplified method for quantitative determination of lipid in a sample of lipid aggregate which is met in study of biomembrane containing lipid and in confirmation of composition of lipid-containing membrane synthesized artificially. Lipid is determined quantitatively by treating a sample with a surfactant in an aqueous medium to obtain a liquid sample dispersion, then reacting the dispersion with a reagent containing cupric ion and bicinchoninic acid to reduce the cupric ion to cuprous ion; and measuring a colored state developed by complex formation of bicinchoninic acid with cuprous ion. This method can be effectively applied to determination of amphiphilic lipid having a tendency of gathering in an aqueous medium.

Claims: A method for quantitative determination of lipid, characterized in that said method comprises preparing a liquid sample dispersion by treating a sample containing lipid with a surfactant in an aqueous medium; reacting the liquid sample dispersion with a reagent containing cupric ion and bicinchoninic acid to reduce the cupric ion to cuprous ion; and measuring a colored state developed by complex formation of bicinchoninic acid with cuprous ion. A method according to claim 1, wherein the sample in the aqueous medium contains liposome.

Inventors: KAWAGUCHI MASAHIRO, NOMOTO TSUYOSHI, OHYAMA JUNJI, TOMIDA YASUKO, YAMAMOTO NOBUKO, KAWAGUCHI, MASAHIRO, NOMOTO, TSUYOSHI, OHYAMA, JUNJI, TOMIDA, YASUKO, YAMAMOTO, NOBUKO
Applicants: CANON KK, CANON KABUSHIKI KAISHA
Application year: 1992
Main classification: G01N33
IPC codes: G01N33