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[223] Assay of coenzyme Q10 in blood
[223]
179
ASSAY OF COENZYME Qlo IN BLOOD
Procedure 2. Urines of adult males (520 ml) are concentrated to 70 ml using a deep-freeze dryer. To the concentrated urine is added an equal volume of 3 N hydrochloric acid, and the mixture is kept at 75 ° for 1.5 hours. The reaction mixture is extracted with three 35-ml portions of ethyl acetate. The combined extracts are washed with water saturated with sodium sulfate and dried over anhydrous sodium sulfate. The resulting solution is evaporated to dryness to obtain a brown residue (162 mg). The residue is dissolved in chloroform, and the insoluble matters are filtered off. The filtrate is made up to a volume of 1 ml for carrying out the TLC. The solution (0.5 ml) is applied on a silica gel plate, developed with cyclohexane-ether-ethanol (3:1:1) and extracted with ether as described in Procedure i under Extraction. The resulting Q-lactone fraction is dissolved in petroleum ether (1 ml) and stirred with Lindler catalyst (10 mg) for 1 hour in an atmosphere of hydrogen, giving a colorless solution. To the reaction mixture is added a solution of bis(trimethylsilyl)acetamide (0.2 ml), and the mixture is stirred for 30 minutes under hydrogen. After removal of the catalyst, the filtrate is evaporated to dryness at 45 ° under reduced pressure at 15 mm Hg and then 0.02 nnn Hg. The residue is dissolved in carbon tetrachloride and made up to a volume of 1 ml to be submitted for the GLC. The GLC is carried out under the conditions given in Table V. The peak of Q-lactone is obtained at 20.8 minutes. The quantitative analysis can be carried out in the similar manner to that described for the procedure in the section on assay. TABLE V CONDITIONS FOR GAS CHROMATOGRAPHY Carrier gas, N~
Flow rate of detector
Temperature (°C) Column
Evaporator
Inlet pressure (kg/cm2)
Flow rate (ml/min)
Hydrogen (ml/min)
Air (ml/min)
220
245
1.1
50
50
500
[ 2 2 3 ] A s s a y of C o e n z y m e Q l o in B l o o d
By ELLIOT REDALIEU a n d KARL FOLKERS Principle. This assay is based on the reaction between ethyl cyanoacetate and coenzyme Q10 (CoQ10) in a basic medium, and the determina-
180
UBIQUINONE
[223]
GROUP
o
O-
I
O C H 3 0 ~ CH30"
y
C2HsOC
c-- CH3 _
CH3
CH30" T
"R
O (1)
H
O
C2HsOC
\
CH30~ ~
"R
o
O-
(11)
(111)
CIt30~
CI130~CH3
CH'3
No,U I
"R
O-
O
c
O
o
C~HsOC
[I
O-
-R O
o (IV)
(V) CH3
I
R = (CH2CH~CCH2)mH
tion of the ions, (II) and (IV), which form and provide the blue color) A method for the purification of coenzyme Qx0 from blood and the colorimetric assay has been found to be reproducible to the extent of 93 -46% in the range of 2-25/~g~ of coenzyme Q10. Me~od Reagents. All solvents are of reagent grade.
n-Hexane and ethyl cyanoacetate (Eastman Organic Chemicals), redistilled Triton B (N-benzyltrimethylammonium hydroxide), commercially available from the Miles Chemical Company as a 40% solution in methanol. The solvent from 17 ml of this solution was removed in vacuo at bath temperatures below 45 °. Heating above this temperature causes undesirable chemical changes leading to a final yellow solution. The dry residue which remains is dissolved i E. Redalieu, I. M. Nilsson,T. M. Farley,K. Folkers,and F. R. Koniuszy, Anal Biochem. 23, 132 (1968). sE. Redalieu, I. M. Nilsson,J. L. G. Nilsson,D. L. Kjaer-Pedersen,and Karl Folkers, Intern. Y. Vitamin Res. 38, 345 (1968).
[223]
ASSAY OF COENZYME Q10 IN BLOOD
181
in 50 ml of absolute ethanol and the solution filtered through Celite before use. This solution of Triton B in ethanol can be used for approximately 1 week, but should be stored in a tightly stoppered bottle and again filtered through Celite when it becomes turbid.
Extraction of Coenzyme Qlo from Blood. One hundred milliliters of acetone is added to 5.0 ml of blood in an 8-ounce bottle with a screw-cap, and the mixture is shaken overnight. To prevent leakage during shaking, a screw-cap with a polyseal liner is used on the bottle. The mixture is then suction-filtered through a coarse porosity sintered-glass filter (ASTM 40-60, c). The sintered glass filter can be replaced by a Bfichner funnel that is prepared with a pad of Celite. The filtrate is collected in a 250-mi round-bottomed flask. The bottle is washed twice with 25-ml portions of benzene, and the washings are passed through the filter and collected. The combined acetone-benzene filtrate is concentrated at reduced pressure on a rotary evaporator with the evaporating flask immersed in a water bath at 40°-50 °. If the temperature exceeds 50 °, the recovery of CoQ10 is decreased. Residual water is removed from the residue by adding 75-100 ml of a 50:50 mixture of benzene and absolute alcohol and concentrating again in vacuo with the evaporating flask at 40°-50 °. The residue is purified by thin-layer chromatography (TLC). Purification of Coenzyme Q~o by TLC. The residue containing the coenzyme Q10 which was extracted from the blood sample is purified by thin-layer chromatography on a 1-mm thick silica gel G plate (20 X 20 cm). The sample is applied to the plate in acetone solution as a streak, and the plate is then developed in a 40:60 ether-n-hexane mixture along with a reference spot of pure CoQ~0 (R/ 6.5-8.0). The ether-n-hexane system for TLC can be replaced by a 1:1 chloroform-benzene system. The area opposite the reference spot is scraped and eluted with 15-20 ml of anhydrous ether. The solvent is evaporated from the eluate under a stream of nitrogen, and the colorimetric determination is performed on the dry residue. Colorimetric Determination of Coenzyme Q~o. Exactly 100 gl of absolute ethanol and 50 gl of ethyl cyanoacetate are added to the residue from chromatography. After addition of 50 gl of 0.8 N Triton B in absolute ethanol, the solution is transferred to the cuvette of a Beckman-Spinco Model 151 spectrocolorimeter. The color that develops is read at 630 nm at 30-second intervals against a reagent blank. The color reaches a maximum intensity in 3-5 minutes. The amount of CoQ10 which is present is determined by comparing the maximum intensity to a standard curve. The standard curve is prepared by determining the maximum intensities of 2.5, 5.0, 7.5, 10, and 20 gg of pure coenzyme Q~0.
179
ASSAY OF COENZYME Qlo IN BLOOD
Procedure 2. Urines of adult males (520 ml) are concentrated to 70 ml using a deep-freeze dryer. To the concentrated urine is added an equal volume of 3 N hydrochloric acid, and the mixture is kept at 75 ° for 1.5 hours. The reaction mixture is extracted with three 35-ml portions of ethyl acetate. The combined extracts are washed with water saturated with sodium sulfate and dried over anhydrous sodium sulfate. The resulting solution is evaporated to dryness to obtain a brown residue (162 mg). The residue is dissolved in chloroform, and the insoluble matters are filtered off. The filtrate is made up to a volume of 1 ml for carrying out the TLC. The solution (0.5 ml) is applied on a silica gel plate, developed with cyclohexane-ether-ethanol (3:1:1) and extracted with ether as described in Procedure i under Extraction. The resulting Q-lactone fraction is dissolved in petroleum ether (1 ml) and stirred with Lindler catalyst (10 mg) for 1 hour in an atmosphere of hydrogen, giving a colorless solution. To the reaction mixture is added a solution of bis(trimethylsilyl)acetamide (0.2 ml), and the mixture is stirred for 30 minutes under hydrogen. After removal of the catalyst, the filtrate is evaporated to dryness at 45 ° under reduced pressure at 15 mm Hg and then 0.02 nnn Hg. The residue is dissolved in carbon tetrachloride and made up to a volume of 1 ml to be submitted for the GLC. The GLC is carried out under the conditions given in Table V. The peak of Q-lactone is obtained at 20.8 minutes. The quantitative analysis can be carried out in the similar manner to that described for the procedure in the section on assay. TABLE V CONDITIONS FOR GAS CHROMATOGRAPHY Carrier gas, N~
Flow rate of detector
Temperature (°C) Column
Evaporator
Inlet pressure (kg/cm2)
Flow rate (ml/min)
Hydrogen (ml/min)
Air (ml/min)
220
245
1.1
50
50
500
[ 2 2 3 ] A s s a y of C o e n z y m e Q l o in B l o o d
By ELLIOT REDALIEU a n d KARL FOLKERS Principle. This assay is based on the reaction between ethyl cyanoacetate and coenzyme Q10 (CoQ10) in a basic medium, and the determina-
180
UBIQUINONE
[223]
GROUP
o
O-
I
O C H 3 0 ~ CH30"
y
C2HsOC
c-- CH3 _
CH3
CH30" T
"R
O (1)
H
O
C2HsOC
\
CH30~ ~
"R
o
O-
(11)
(111)
CIt30~
CI130~CH3
CH'3
No,U I
"R
O-
O
c
O
o
C~HsOC
[I
O-
-R O
o (IV)
(V) CH3
I
R = (CH2CH~CCH2)mH
tion of the ions, (II) and (IV), which form and provide the blue color) A method for the purification of coenzyme Qx0 from blood and the colorimetric assay has been found to be reproducible to the extent of 93 -46% in the range of 2-25/~g~ of coenzyme Q10. Me~od Reagents. All solvents are of reagent grade.
n-Hexane and ethyl cyanoacetate (Eastman Organic Chemicals), redistilled Triton B (N-benzyltrimethylammonium hydroxide), commercially available from the Miles Chemical Company as a 40% solution in methanol. The solvent from 17 ml of this solution was removed in vacuo at bath temperatures below 45 °. Heating above this temperature causes undesirable chemical changes leading to a final yellow solution. The dry residue which remains is dissolved i E. Redalieu, I. M. Nilsson,T. M. Farley,K. Folkers,and F. R. Koniuszy, Anal Biochem. 23, 132 (1968). sE. Redalieu, I. M. Nilsson,J. L. G. Nilsson,D. L. Kjaer-Pedersen,and Karl Folkers, Intern. Y. Vitamin Res. 38, 345 (1968).
[223]
ASSAY OF COENZYME Q10 IN BLOOD
181
in 50 ml of absolute ethanol and the solution filtered through Celite before use. This solution of Triton B in ethanol can be used for approximately 1 week, but should be stored in a tightly stoppered bottle and again filtered through Celite when it becomes turbid.
Extraction of Coenzyme Qlo from Blood. One hundred milliliters of acetone is added to 5.0 ml of blood in an 8-ounce bottle with a screw-cap, and the mixture is shaken overnight. To prevent leakage during shaking, a screw-cap with a polyseal liner is used on the bottle. The mixture is then suction-filtered through a coarse porosity sintered-glass filter (ASTM 40-60, c). The sintered glass filter can be replaced by a Bfichner funnel that is prepared with a pad of Celite. The filtrate is collected in a 250-mi round-bottomed flask. The bottle is washed twice with 25-ml portions of benzene, and the washings are passed through the filter and collected. The combined acetone-benzene filtrate is concentrated at reduced pressure on a rotary evaporator with the evaporating flask immersed in a water bath at 40°-50 °. If the temperature exceeds 50 °, the recovery of CoQ10 is decreased. Residual water is removed from the residue by adding 75-100 ml of a 50:50 mixture of benzene and absolute alcohol and concentrating again in vacuo with the evaporating flask at 40°-50 °. The residue is purified by thin-layer chromatography (TLC). Purification of Coenzyme Q~o by TLC. The residue containing the coenzyme Q10 which was extracted from the blood sample is purified by thin-layer chromatography on a 1-mm thick silica gel G plate (20 X 20 cm). The sample is applied to the plate in acetone solution as a streak, and the plate is then developed in a 40:60 ether-n-hexane mixture along with a reference spot of pure CoQ~0 (R/ 6.5-8.0). The ether-n-hexane system for TLC can be replaced by a 1:1 chloroform-benzene system. The area opposite the reference spot is scraped and eluted with 15-20 ml of anhydrous ether. The solvent is evaporated from the eluate under a stream of nitrogen, and the colorimetric determination is performed on the dry residue. Colorimetric Determination of Coenzyme Q~o. Exactly 100 gl of absolute ethanol and 50 gl of ethyl cyanoacetate are added to the residue from chromatography. After addition of 50 gl of 0.8 N Triton B in absolute ethanol, the solution is transferred to the cuvette of a Beckman-Spinco Model 151 spectrocolorimeter. The color that develops is read at 630 nm at 30-second intervals against a reagent blank. The color reaches a maximum intensity in 3-5 minutes. The amount of CoQ10 which is present is determined by comparing the maximum intensity to a standard curve. The standard curve is prepared by determining the maximum intensities of 2.5, 5.0, 7.5, 10, and 20 gg of pure coenzyme Q~0.