WO2019045079A1 - Méthode de mesure de la capacité d'efflux de cholestérol - Google Patents

Méthode de mesure de la capacité d'efflux de cholestérol Download PDF

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WO2019045079A1
WO2019045079A1 PCT/JP2018/032500 JP2018032500W WO2019045079A1 WO 2019045079 A1 WO2019045079 A1 WO 2019045079A1 JP 2018032500 W JP2018032500 W JP 2018032500W WO 2019045079 A1 WO2019045079 A1 WO 2019045079A1
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cholesterol
cells
sample
cell
measurement
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Japanese (ja)
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知 清水
智之 臼井
宮崎　修
吉田　博
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積水メディカル株式会社
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Priority to JP2022210923A priority patent/JP7474448B2/ja

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

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  • the present invention relates to a method of measuring the ability to extract cholesterol.
  • the invention includes those described below.
  • [1] a step of adding stable isotope-labeled cholesterol to a culture solution containing cells and culturing them, and causing the cells to incorporate the stable isotope-labeled cholesterol; (2) culturing the cholesterol-incorporated cells in the presence of a test substance, (3) measuring the amount of stable isotope-labeled cholesterol extracted from the cells; A method for measuring the ability of the test substance to extract cholesterol, including [2] The method according to [1], wherein in the above-mentioned (1), the concentration of the stable isotope-labeled cholesterol in the culture solution containing the cells is 2 ⁇ g / mL to 30 ⁇ g / mL.
  • [3] The method according to [1] or [2], wherein the culture time is shorter than 48 hours in (1).
  • [4] The method according to any one of [1] to [3], wherein in the above (1), the concentration of cells in the culture solution is higher than 2.0 ⁇ 10 5 cells / mL.
  • a cholesterol labeled with a stable isotope other than the stable isotope is added at a predetermined concentration to the culture obtained in (2) above, the amount is measured, and a calibration curve is prepared.
  • the method according to any one of [1] to [4], further comprising [6] The method according to any one of [1] to [5], wherein the measurement of the amount of stable isotope-labeled cholesterol is quantification by mass spectrometry.
  • the test substance is a serum sample, a high density lipoprotein or apolipoprotein A-1 separated from serum, or a serum sample from which apolipoprotein B has been removed, according to any one of [1] to [6] the method of.
  • a method of measuring the ability to extract cholesterol by measuring increase or decrease in the amount of cholesterol by mass spectrometry using stable isotope-labeled cholesterol which can satisfy clinical requirements.
  • Cell count results Cholesterol uptake ability. Measurement results of cell viability. The measurement result of the amount of cholesterol in the culture supernatant by mass spectrometry.
  • CAD coronary artery disease
  • statin HMG-CoA reductase inhibitor
  • RCT reverse cholesterol transfer system
  • the first step in the cholesterol reverse transfer system is cholesterol withdrawal from cells (c-efflux), which is said to be the most important function of HDL. It is reported that c-efflux ability and CAD onset rate by serum HDL are negatively correlated (Khera, et al. N Engl J Med 2011 364 127).
  • the c-efflux capacity of serum HDL is currently measured using cholesterol labeled with a radioactive isotope such as 3 H.
  • a radioactive isotope such as 3 H.
  • a radioisotope-free assay system would be required to evaluate c-efflux ability at the clinical examination site, and examined the c-efflux ability evaluation system using stable isotopes.
  • the mouse macrophage cell line J774 was seeded at a density of 3 ⁇ 10 5 cells / well in a 24-well plate and incubated for 24 hours in the presence of 5 ⁇ g / well 2 H 6 -cholesterol (37 ° C., 5% CO 2 ). After incubation with 0.3 mmol / L cAMP for 18 hours to activate ABCAl, 0.25% to 2% apo B-depleted serum was added and incubated for 2 hours. Cholesterol was extracted from the supernatant by the Folch method, and 2 H 6 -cholesterol was quantified using Q-TOF MS (Maxis 3G; Bruker). As a result, it was confirmed that the apo B removal serum concentration-dependent increase in the amount of 2 H 6 -cholesterol.
  • the method of the present invention has a good correlation with the results of measurement methods using conventional radioactive isotopes. Moreover, the method of the present invention is excellent in simultaneous reproducibility and daily reproducibility. Furthermore, the measurement range is wide.
  • radioisotopes and fluorescent dyes have been used as cholesterol labeling substances in cholesterol withdrawal ability measurement, and withdrawal ability was evaluated using measured values of radioactivity and fluorescence intensity.
  • radioactive isotopes and fluorescent dyes are measured without being distinguished from those in which cholesterol is labeled, even when the labeling substance is released from cholesterol (labeling substance alone). Therefore, the conventional method can not deny the possibility of measuring released radioactive isotopes and fluorescent dyes, and thus has not been a highly accurate and reliable cholesterol measuring method.
  • radioactive isotope or fluorescent dye to be labeled is changed as cholesterol to be incorporated into cells to distinguish free cholesterol from cholesterol ester, the extracted free cholesterol is metabolized by cholesterol in the conventional method. It was not possible to identify and track when it became an ester. Furthermore, the relationship between the radioactivity of the labeling substance and the intensity of the fluorescence intensity and the amount of labeled cholesterol (referred to as specific activity etc.) was measured because it differs for each labeled cholesterol used in the test. The evaluation of the ability to withdraw cholesterol could only be a relative one.
  • labeling cholesterol with a radioactive isotope, a fluorescent dye, or a stable isotope may be simply referred to as a radioactive isotope label, a fluorescent label, or a stable isotope label.
  • cholesterol in order to measure stable isotope labeled cholesterol by mass spectrometry, cholesterol can be accurately measured based on structural information.
  • it is possible to distinguish and measure free cholesterol and cholesterol ester it is also possible to track the metabolic state of cholesterol after being withdrawn from cells.
  • Example 1 Selection of Culture Conditions for Cholesterol Uptake (1)
  • (1-1) Cholesterol uptake (1) Add DMEM (manufactured by GIBCO) (hereinafter referred to as 10% FBS-DMEM) added FBS (manufactured by BIOLOGICAL INDUSTRIES, 04-001-1A) to a concentration of 10%, and add FBS to a concentration of 1% J774 cells were added to each culture medium of each of the following culture media (hereinafter referred to as 1% FBS-DMEM) and DMEM supplemented with BSA to a concentration of 0.2% (hereinafter referred to as 0.2% BSA-DMEM).
  • 1% FBS-DMEM DMEM
  • BSA-DMEM DMEM supplemented with BSA to a concentration of 0.2%
  • the cells were seeded at a concentration of 3 ⁇ 10 5 cells / mL, 2.0 ⁇ 10 5 cells / mL, and 6.0 ⁇ 10 5 cells / mL.
  • cholesterol (2,2,3,4,4,6) -D6 (Kanto Kagaku Co., Ltd., 49123-87) dissolved in ethanol at a concentration of 10 mg / mL is added, and the cholesterol final concentration is 50 ⁇ g / mL.
  • Cell dilutions of 10 ⁇ g / mL and 2 ⁇ g / mL were prepared.
  • Sandoz 58-035 (Sigma, S9318), which is an ACAT (acyl-CoA: cholesterol acyltransferase) inhibitor, was added to all the samples to a concentration of 2 ⁇ g / mL. After 0.5 mL of cell dilution prepared under each condition was seeded on a 24-well culture plate and cultured for 24 hours, 48 hours, 72 hours, 96 hours, all cell culture supernatants were removed and washed twice with PBS . Each culture condition is shown in Table 1.
  • the collected cell solution 1 and the collected cell solution 2 were pooled respectively to make a total of 500 ⁇ L. Subsequently, the resultant was centrifuged at 12,000 rpm and 4 ° C. for 10 minutes, and 25 ⁇ L of supernatant was collected from each sample and dried by a centrifugal evaporator to obtain a dried sample.
  • the flow rate was 0.3 mL / min
  • the column temperature was 40 ° C.
  • a solution of acetonitrile and methanol mixed at 4: 1 was used as a mobile phase for isocratic elution.
  • APCI method was used for mass spectrometry.
  • For each sample prepare a calibration curve from the peak area of cholesterol (2,2,3,4,4,6) -D6 diluted to concentrations of 10, 5, 2 and 1 ng / mL, respectively, per well. It was calculated as the amount of cellular cholesterol contained in The measurements (1-2) to (1-4) were not performed on the cultures prepared in (1-1) above that were judged to be poor in cell growth or overgrowth.
  • Test results (1) The results of “Measurement of cell number” of (1-2) above are shown in FIG. 1, and the results of “Measurement of cholesterol in cells by mass spectrometry” of (1-4) above are shown in FIG.
  • the dilution concentration of J774 cells is 6.0 ⁇ 10 5 cells / mL
  • the culture solution is 10% FBS-DMEM
  • the culture time is 24 hours
  • the number of cells after cholesterol uptake is the largest.
  • the cell growth was shown to be good.
  • the cells did not grow in 0.2% BSA-DMEM (data not shown).
  • FIG. 2 it was shown that the cholesterol uptake amount is the best under the same conditions.
  • Example 2 Selection of culture conditions for cholesterol uptake (2) (2-1) Cholesterol uptake (2) Cholesterol (2,2,3,4,4,6) -D6 at a concentration of 10 mg / mL in ethanol diluted with 10% FBS-DMEM to a concentration of 6.0 ⁇ 10 5 cells / mL The solution was added to the solution to prepare cell dilutions of a final cholesterol concentration of 50 ⁇ g / mL, 20 ⁇ g / mL, 10 ⁇ g / mL, and 5 ⁇ g / mL, respectively. In addition, Sandoz 58-035 was added to all samples to a concentration of 2 ⁇ g / mL. The cell dilution of each condition was seeded at 0.5 mL each on a 24-well culture plate and cultured for 24 hours.
  • Example 3 Measurement of cholesterol withdrawal ability by serum sample (3-1) Preparation of apo B-depleted serum sample (1) A solution of PEG6000 (manufactured by Wako Pure Chemical Industries, Ltd., 169-09125) in 200 mM Glycine pH 7.4 at a concentration of 20% (w / v) is added to 200 ⁇ L of serum samples (3 types; samples 1, 2, 3) 80 ⁇ L was added and incubated for 20 minutes at room temperature. Then, the supernatant was recovered by centrifugation at 10,000 rpm for 30 minutes at 4 ° C. to prepare an apoB-depleted serum sample.
  • PEG6000 manufactured by Wako Pure Chemical Industries, Ltd., 169-09125
  • any of the three apo B-depleted serum specimens obtained in the above (3-1) “Preparation of apo B-depleted serum specimen (1)”
  • the mixture was diluted with 0.2% BSA-DMEM to 2.0%, 1.0%, 0.5% and 0.25% concentrations, respectively, and 0.25 mL each was added and cultured for 2 hours. The culture supernatant was collected.
  • the measurement value of each sample obtained from the culture supernatant is the following using the measurement value of the internal standard sample: Calculated from the correction equation of Measurement of the internal standard sample, 20,10,5,2,1Ng amount of cholesterol (3,4) - 13 was calculated by a calibration curve from the peak area of C 2.
  • Test results (3) The result of the above (3-5) “measurement of the amount of cholesterol in the culture supernatant by mass spectrometry” is shown in FIG. According to FIG. 4, the measured values of cholesterol (2,2,3,4,4,6) -D6 increase for all three apo B-depleted serum specimens depending on the addition concentration thereof. This is an increase in the amount of cholesterol (2,2,3,4,4,6) -D6 drawn into the culture supernatant depending on the amount of HDL contained in the added apo B-depleted serum sample. It is shown that. That is, it became clear by this test method that quantitative evaluation of cholesterol withdrawal ability by HDL is possible.
  • Example 4 Effect of hydrolysis treatment on cholesterol measurement (4-1) Extraction of cholesterol by serum sample (2) Using a serum sample (13 types), an apoB-free serum sample was prepared in the same manner as the above (3-1) “Preparation of apoB-depleted serum sample (1)”. Procedure similar to (3-2) “Cholesterol uptake (3)” and (3-3) “cholesterol withdrawal by serum sample” but using serum sample diluted to a concentration of 2.8% Then, a cell culture was obtained using cholesterol (25, 26, 26, 26, 27, 27, 27) -D7 (Kanto Chemical Co., 49123-89) as cholesterol. All culture supernatants were recovered from the culture.
  • the measured values of the internal standard sample were calculated by preparing a calibration curve from peak areas of 20, 10, 5, 2 and 1 ng of cholesterol (25, 26, 26, 26) -D4.
  • the following correction formula is an internal standard sample (cholesterol (25, 26, 26, 26) in a dried sample from 2/3 of the hexane layer (upper layer) prepared in (4-3) and (4-4) above. -Based on the assumption that D4) is included at 20 ng (30 ng ⁇ 2/3).
  • Amount of cholesterol (ng) in 30 ⁇ L of culture supernatant (or cell lysate) sample C x [20 (ng) / D] x 3/2
  • C Cholesterol (25, 26, 26, 26, 27, 27, 27) in the dry sample obtained from the culture supernatant (or cell lysate)-measured value of D7 (ng)
  • D Measured value (ng) of internal standard sample (cholesterol (25, 26, 26, 26)-D4)
  • the total amount of cholesterol (culture supernatant measurement value G) extracted in the culture supernatant per well, and per well after cholesterol extraction was calculated according to the following formula.
  • the following formula is based on 250 ⁇ L of culture supernatant of cells per well (above (4-1)) and 200 ⁇ L of 1% sodium cholate (above (4-2)) added per well. .
  • G E ⁇ 250 ( ⁇ L) / 30 ( ⁇ L) [Cell measurement value]
  • H F ⁇ 200 ( ⁇ L) / 30 ( ⁇ L)
  • E Amount of cholesterol in 30 ⁇ L of culture supernatant sample (ng)
  • F amount of cholesterol in 30 ⁇ L of cell lysate sample (ng)
  • c-efflux value (%) (G: culture supernatant measurement value) / ⁇ (G: culture supernatant measurement value) + (H: cell measurement value) ⁇
  • FIGS. 5 and 6 show the results of the c-efflux value determined in the above (4-5) “Measurement of the amount of cholesterol in the culture supernatant and cells by mass spectrometry (2)” are shown in FIGS. 5 and 6.
  • FIG. 5 shows the c-efflux value of the sample obtained in the above (4-3) “cholesterol extraction (with hydrolysis) (1)” by X, the above (4-4) “cholesterol extraction (without hydrolysis) (1) The plot of the measured value of each sample is shown, where Y is the c-efflux value of the sample obtained in 2.).
  • FIG. 6 shows a comparison of the c-efflux values obtained for “with hydrolysis” and “without hydrolysis” for 4 of the 13 tested samples (samples 4, 5, 6, 7) .
  • Example 5 Correction effect by internal standard on cholesterol measurement (5-1) Cholesterol withdrawal by serum sample (3) Using serum samples (3 types; samples 5-1, 5-2, and 5-3), apo B by the same procedure as (3-1) “Preparation of apo B-depleted serum test (1)” above. Depleted serum samples were prepared. Using this serum sample, a cell culture was obtained by the same procedure as the above (4-1). All culture supernatants were recovered from the culture. The remaining cells were treated in the same manner as in (4-2) "Preparation of cell lysate (1)" to prepare a cell lysate.
  • Example 6 Effects of added concentrations of apoA-1 and apoB-depleted serum on cholesterol measurement (6-1) Cholesterol withdrawal by serum samples (4) Using a serum sample (one type), an apoB-free serum sample was prepared in the same manner as the above (3-1) "Preparation of apoB-depleted serum sample (1)".
  • apo B-depleted serum specimen is 0.2% Diluted with BSA-DMEM to four concentrations of 5.6%, 2.8%, 1.4% and 0.7%, and purified apo A-1 (Alfa Aesar, J64506) at 16 ⁇ g / mL, 8 ⁇ g / mL Cell cultures were obtained using each diluted to 4 concentrations of 4 ⁇ g / mL and 2 ⁇ g / mL. All culture supernatants were recovered from the culture. Using the remaining cells, a cell lysate was prepared in the same manner as the above (4-2) “Preparation of cell lysate (1)”.
  • Example 7 Daily reproducibility (7-1) Cholesterol withdrawal by serum samples (5) Using serum samples (two types; samples 7-1 and 7-2), apo B-removed serum sample is prepared in the same procedure as (3-1) “Preparation of apo B-removed serum sample (1)” above. Prepared. Using this serum sample, a cell culture was obtained by the same procedure as the above (4-1). All culture supernatants were recovered from the culture. The remaining cells were treated in the same manner as in (4-2) "Preparation of cell lysate (1)" to prepare a cell lysate.
  • Example 8 Correlation between measurement method using radioisotope-labeled cholesterol and the method of the present invention (8-1) Preparation of apo B-depleted serum sample Using serum samples (8 types) having normal lipid value Then, an apoB-depleted serum sample was prepared in the same manner as the above (3-1) “Preparation of apoB-depleted serum sample (1)”.
  • the collected cell solution 1 and the collected cell solution 2 were respectively pooled to form a total volume of 500 ⁇ L and centrifuged at 12,000 rpm and 4 ° C. for 10 minutes, and 100 ⁇ L of supernatant was collected from each sample to obtain a cell extraction sample.
  • c-efflux value (%) (O: culture supernatant measurement value) / ⁇ (O: culture supernatant measurement value) + (P: cell measurement value) ⁇
  • Test results (8) The relationship between c-efflux values obtained in the above tests (8-5) and (8-6) is shown in FIG. FIG. 10 plots each sample with the c-efflux value of each sample obtained in (8-5) as X and the c-efflux value of each sample obtained in (8-6) as Y. ing.
  • Example 9 Correlation between measurement method using fluorescently labeled cholesterol and the method of the present invention (9-1) Preparation of apo B-depleted serum sample Using serum samples (8 types) having normal lipid value ApoB-depleted serum specimen was prepared in the same manner as (3-1) “Preparation of apoB-depleted serum specimen (1)” above.
  • c-efflux value (%) (S: culture supernatant measurement value) / ⁇ (S: culture supernatant measurement value) + (T: cell measurement value) ⁇
  • Test results (9) The relationship between c-efflux values obtained in the above tests (9-5) and (9-6) is shown in FIG. FIG. 11 plots each sample with the c-efflux value of each sample obtained in the above (9-5) as X and the c-efflux value of each sample obtained in the above (9-6) as Y. .
  • Example 10 Correlation between measurement methods by measuring 41 samples (10-1) Preparation of apo B-depleted serum sample Using serum samples (41 types) having a normal lipid value, the above (3-1) “ The apoB-depleted serum sample was prepared in the same manner as in “Preparation of apoB-depleted serum sample (1)”.
  • the total amount of cholesterol (measurement value of culture supernatant) drawn in the culture supernatant per well, and per cholesterol before cholesterol withdrawal was calculated by the following formula.
  • the following formula is 250 ⁇ L of culture supernatant of cells per well (above (10-2-2)), and 200 ⁇ L of 1% sodium cholate added per well (above (10-2-3)) Based on a certain thing.
  • the DPM value of the cholesterol component drawn out in the culture supernatant per well according to the following formula (the culture supernatant measurement value), and it is taken into cells per well before cholesterol withdrawal.
  • the DPM value (cell measurement value) of cholesterol was calculated respectively.
  • the cells were washed twice with 10 mM HEPES-MEM (pH 7.4), and 200 ⁇ L each of 1% sodium cholate was added and incubated for 4 hours at room temperature (ie, cell lysate using non-Actor-supplemented cells) Prepared).
  • the fluorescence intensity of the cholesterol component extracted in the culture supernatant per well according to the following formula (culture supernatant measurement value), and is taken up in the cells per well before cholesterol withdrawal
  • the fluorescence intensity (cell measurement value) of cholesterol was calculated respectively.
  • FIG. 12A is a plot of the c-efflux value (X axis) obtained in (10-3) and the c-efflux value (Y axis) obtained in (10-2) for the 41 samples.
  • FIG. 12B shows plots of the c-efflux value (X axis) obtained in (10-4) above and the c-eflux value (Y axis) obtained in (10-2) above.
  • FIG. 12C shows plots of the c-efflux value (X axis) obtained in (10-3) above and the c-efflux value (Y axis) obtained in (10-4) above .
  • the method of the present invention is useful as an alternative to the method using radioactive isotopes, which can also be used in a clinical laboratory or research facility where radioactive isotopes can not be used.
  • the method of the present invention is expected to be greatly useful for studies on HDL metabolism and function.

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Abstract

L'invention concerne une méthode de mesure de la capacité d'efflux de cholestérol qui peut satisfaire des exigences cliniques. La méthode de mesure de la capacité d'efflux de cholestérol d'une substance d'essai comprend : (1) une étape d'ajout d'un cholestérol marqué par un isotope stable à un milieu de culture contenant des cellules et la culture pour provoquer l'absorption du cholestérol marqué par un isotope stable par les cellules ; (2) une étape de culture, en présence d'une substance d'essai, des cellules qui ont absorbé le cholestérol ; et (3) une étape de mesure de la quantité de cholestérol marqué par un isotope stable excrété par les cellules.
PCT/JP2018/032500 2017-08-31 2018-08-31 Méthode de mesure de la capacité d'efflux de cholestérol WO2019045079A1 (fr)

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