Quantitative determination of lipids

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-aminoantipyrine 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 US-A-4 839 294 discloses an enzyme-free, non-hydrolizing 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 characteristic color to the lipid-contaiing fluid. 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. The GB-A-2 043 244 discloses a process for the determination of two or more components in a liquid sample. In the process, reagents for the different components are added to the sample, with the composition of the reagents so chosen that the separate components for different determinations do not interfere with one another. Absorptions of the respective reaction products are measured in the sample at two different wavelengths. 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. 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. According to the present invention, there is provided a method for quantitatively determining lipid, comprising preparing a liquid sample dispersion by treating a sample containing lipid with a surfactant in an aqueous medium to form a micelle composed of the liquid and the surfactant, the surfactant exhibiting no absorption in the UV region; and measuring light absorbance of the liquid sample dispersion in an ultraviolet region. DESCRIPTION OF THE PREFERRED EMBODIMENT Firstly, the method of determining lipid in the present invention is described below. In the lipid determination method 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, with a surfactant exhibiting no UV absorption in the measured UV range, the UV absorption is caused only by the lipid in a lipid-surfactant micelle state and the absorbance depends only on the concentration of the lipid, thus the lipid concentration being determined by measuring the UV absorbance. Any wavelength may be selected for the absorbance measurement within the range of from 230 to 290 nm if any substance other than lipid does not exist. However, if existence of other substance is anticipated, the wavelength needs to be selected at which the coexisting substance exhibits no or little absorbance. In research fields of handling or mimicking a biomembrane, the anticipated coexisting substance in most cases is protein. In this circumstance, the wavelength is preferably selected at around 240 nm, avoiding the maximum absorption of the protein of 280 nm. Any surfactant may be used which exhibits no absorption in the UV region. The example of the useful surfactant includes octylglucoside (n-octyl-β-D-glucopyranoside), CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and HECAMEG (6-O-(N-heptylcarbamoyl)-methyl-a-D-glucopyranoside). 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. The present invention is described in more detail by reference to Examples. Example 1 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 an 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. Example 2 A liquid liposome dispersion was prepared in the same manner as in 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 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. 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.

Claims: A method for quantitative determination of lipid, comprising: preparing a liquid sample dispersion by treating a sample containing lipid with a surfactant in an aqueous medium to form a micelle composed of the lipid and the surfactant, the surfactant exhibiting no absorption in the UV region; and measuring light absorbance of the liquid sample dispersion in an ultraviolet region. A method according to claim 1, wherein the absorption is measured at 240 nm. A method according to claim 1, wherein the sample contains liposome.

Inventors: KAWAGUCHI MASAHIRO, NOMOTO TSUYOSHI, OHYAMA JUNJI, TOMIDA YASUKO, YAMAMOTO NOBUKO, KAWAGUCHI, MASAHIRO, NOMOTO, TSUYOSHI, OHYAMA, JUNJI, TOMIDA, YASUKO, YAMAMOTO, NOBUKO, KAWAGUCHI, MASAHIRO, C/O CANON KABUSHIKI KAISHA, Nomoto, Tsuyoshi, c/o Canon Kabushiki Kaisha, Ohyama, Junji, c/o Canon Kabushiki Kaisha, Tomida, Yasuko, c/o Canon Kabushiki Kaisha, YAMAMOTO, NOBUKO, C/O CANON KABUSHIKI KAISHA
Applicants: CANON KK, CANON KABUSHIKI KAISHA
Application year: 1992
Main classification: G01N33
IPC codes: G01N33