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Coenzyme Q. XIII. Isolation, assay and human urinary levels of coenzyme Q10
ARCHIVES
OF
BIOCHEMISTRY
Coenzyme
AND
Q.
XIII.
87,
BIOPHYSICS
Isolation,
29&305-(1960)
Assay
and
of Coenzyme FRANK
R. KONIUSZY,
Merck,
Sharp
Human
Research
Received
Laboratories, October
Levels
Q,,
PAUL H. GALE, ATWOOD AND KARL FOLKERS
& Dohme
Urinary
Rahway,
C. PAGE,
New
JR.
Jersey
21, 1959
Pure coenzyme Qlo has been isolated from normal human male urine; a calorimetric assay for determining QrJevels in urine has been devised and applied to 160 collections representing 63 males and females. The average excretion is ea. 55 pg./24 hr. for males and 22 pg./24 hr. for females. Thirty-five per cent of the 63 individuals excreted
Coenzyme Qlo (I) of the coenzyme Q group (five members, i.e., Q6-Q10), exists widespread in animal species. The existence of coeneyme Qlo in the human body was estab0 CH,O
and subsequently, the amounts of the coenzyme excreted per day by a group of presumably normal individuals were determined. These data are basic for an interpretation of the analytical values of human urines from various disease states for possible guidance of exploratory medical studies. EXPERIMENTAL
CH,O 0
lished by the isolation of the crystalline substance from human heart tissue (1). Recent biochemical reports state that the coenzyme Q group is specific for restoration of cytochrome c reductase activity (2), and for restoration of succinoxidase activity of acetone-extracted mitochondria (3). Mervyn and Morton (4) have reported values, determined spectrophotometrically, of the coenzyme Q content of normal human kidney and 16 specimens of diseased kidney tissue. The content was similar in both cases. It appears that a study of a possible role of coenzyme Q10 in human health and disease would be benefited by data on the blood and urine levels of this substance. Urinary excretion was studied first, and our results are described in this publication. Evidence was sought on whether or not coenzyme Q10 is present in human urine;
AND
RESULTS
RESULTS ON ISOLATION OF COENZYME QIo Coenzyme Q1o was isolated in pure crystalline form from 140 gal. of pooled normal human male urine. It has not been possible to perform quantitative assays on urine itself, but it is possible to assay the material from the first solvent extractives. At this step, the 3.25 g. of extractives contained 16.3 mg. &lo . Crystalline Qlo (10.8 mg.) was obtained from these extractives in 66 % yield. The &lo from urine was identified by its melting point and spectral characterist.ics, including a nuclear magnetic resonance spectrum. It was examined by a radial paper chromatographic system which will separate Qro from its lower homologs (Q9 , etc.). Only Qlo was found in the crystalline product, although a spot with an R/ characteristic of Q9 was detected in the mother liquors from the crystallization. No evidence was ob298
COENZYME
served for the presence, in any of the materials, of additional members of the Q group, nor for metabolic degradation products which retained the substituted benzoquinone nucleus. Experimental A total of 1.40 gal. of normal male urine was collected over a lo-day period. Each day’s collection was processed on the following day. A given day’s collection (12-16 gal.) was concentrated in uucuo at 3(f35” to one-half volume. The concentrate was diluted with an equal volume of denatured ethyl alcohol (2BA). (Methanol should not be eubstituted for ethyl alcohol.) The mixture was then extracted with three portions of Skellysolve B, each equal in volume to the urine concentrate (6-8 gal.). Emulsions, which occasionally formed at this stage, were broken by the addition of a little more alcohol. The combmed Skellysolve extract was washed twice with one-third its volume of water and dried with 100 g. of anhydrous sodium sulfate; it was then concentrated in vacua to yield a thick yellowish gum. From the 140 gal. urine, 3.25 g. of yellow gum was obtained. An aliquot of this gum was assayed calorimetrically and showed the presence of 16.3 mg. coenzyme Q,o in the total residue. The 3.25 g. residue was dissolved in 100 ml. Skellysolve .B, and the solution was chromatographed over a 30 mm. X 12 cm. column of Florisil. The column was washed with 7 1. Skellysolve B, which did not elute a significant amount of Q1,, . The column was then eluted with 12 1. of 10% ethyl ether in Skellysolve B. Concentration of this eluate yielded I.2 g. of yellow gum, containing 12.8 mg. Q10 by c’olorimetric assay. The 1.2 g. gum was dissolved in 15 ml. of 99.37$ methanol which had been equilibrated with isooctane, and the solution was partitioned in a 20 mm. X 50 cm. column containing 100 g. silicic acid which had been silanized (5) by treatment with dimethyldichlorosilane. The partitioning was continued with methanol-isooctane until the visible yellow band was collected separately in approximately 50 mll. eluate. Concentration of this yellow eluate yielded 75 mg. of yellowish white crystalline material which showed 12.3 mg. &lo by assay. Countercurrent distribution of this 75-mg. sample through five plates with 50-ml. portions of 95% dimethylformamide (DMF)-isooctane, to remove some cholesterol, yielded a bright yellow residue from the isooctane layer in the first plate (DMF was the moving phase) that weighed 71.3 mg. This 71.3 mg. residue was dissolved in 30 ml. of absolute methanol. A. few drops of water were added to this solution, and the white precipitate which formed
Q. XIII
299
was removed by centrifugation. The dropwise additions of water and the centrifugings were continued until no more white precipitate formed. The final methanol concentration was approximately 96%. Concentration of the methanol solution to dryness in z)acuo yielded 17.0 mg. of yellow gummy residue, which was dissolved in 1.5 ml. of absolute ethyl alcohol. This solution was diluted with 9 ml. of absolute methanol, and by the following day, 11.9 mg. of yellow crystals had separated; m.p. 42-44”C. The first recrystallization gave crystals melting at 4G-48”; the second, m.p. 4%-49”; the third, m.p. 49-50”; the fourth, m.p. 49-50”. The yield of crystalline coenzyme Q,o was 10.8 mg.
PAPER
STRIP AND NMR DATA Qlo FROM URINE
ox
Samples of the crystalline product, coenzyme &lo from beef heart, and a mixture of the two were papergrammed radially using reversed-phase technique (1). The 32 cm. diameter circular sheet of Whatman No. 1 paper was prepared by passing it once through a 5 % solution of petroleum jelly (Vaseline) in petroleum ether and allowing it to dry in air. A 97:3 mixture of dimethylformamide (Merck, Reagent) and water was the mobile phase. Zones appeared as dark areas when the air-dried papergram was held over an ultraviolet lamp. The crystalline Q from urine, crystalline coenzyme Q10 from beef heart, and a mixture of the two all moved with identical Rr’s in this system, which is readily capable of distinguishing Qlo from other members of the group. The nuclear magnetic resonance spectra of the two crystalline materials were identical.
QUANTITATIVE DETERMINATION IN URINE SAMPLES
OF Qlo
It appeared that the modified Craven’s test, which has been examined (6) with a considerable array of synthetic model quinones and members of the Q group, could be developed into a calorimetric assay for Qlo in urines. The method involves the reaction of &lo and ethyl cyanoacetate in the presence of potassium hydroxide. The blue color which forms is measured after 10 min. A standard curve is shown in Fig. 1. Zero levels (“O”levels) or O-10 pg. signify that there was no
300
KONIUSZY,
GALE,
color; lo-25-pg. levels are estimates based on very weak color reactions; higher levels of the colored Q10 reaction product are measured with more precision. In replicate determinations of the same sample, a standard deviation of f8 pg. was found for any of the levels studied in the lo-lOO+g. range. Greater variation (~15 %) was found in the optical density observed with a given level of Q1o standard on different days. However, all of the levels in the standard in a single assay were altered proportionately. This reaction gives reproducible results with partially purified concentrates from urine samples as well as with crystalline &lo . The established procedure consists of direct extraction of the urine sample and further purification of the extractives by alumina chromatography. Florisil, Decalso, acid-washed alumina, and silicic acid all gave recoveries of over 80%; but the best recoveries (95-100%) were obtained with acid-washed alumina columns. Q10 is decomposed if it is allowed to remain longer than 24 hr. on alumina columns. Since the chromatographic step is completed in about 1 hr., significant losses are avoided. Assay of several aliquots of several collections of pooled urine showed that the varia-
FIG. mination
1. Standard curve for of Coenzyme &IO .
calorimetric
deter-
PAGE
AND
FOLKERS
tion in the assay of a single collection was less than &5%. When pure coenzyme Qio was added to several assayed urine collections, the recoveries of the added QN were 95-100 %. Experimental Procedure. For analysis of the total amount of coenzyme &lo eliminated in human urine over a 24-hr. period, the following procedure was found to be the most convenient and reproducible. The individual urine samples were collected without added preservative. If the determination could be carried out within 1 or 2 days after the final voiding, no detectable loss of &lo was observed. The volume and the pH of the urine were noted. The urine was diluted with an equal volume of denatured alcohol, and this mixture was then shaken vigorously with a volume of Skellysolve B equal to the original volume of urine. The alcoholdiluted urine was separated and discarded. The Skellysolve layer was washed by shaking it with an equal volume of distilled water, separated, then dried by shaking it with 25 g. of anhydrous sodium sulfate. The Skellysolve extract was carefully decanted into a concentration flask without. filtration, since filter paper tends to adsorb Q10 . After the extract was concentrated to dryness in vacua, the residue was dissolved in 10 ml. of Skellysolve B. This solution was chromatographed over a 7 mm. diameter by 12 cm. column of acid-washed alumina. Solvent flow was increased by the application of 2-3 lb./sq. in. air pressure to the top of the column. The chromatogram was washed with 10 ml. of Skellysolve B, and this washing was discarded. The coenzyme Q was eluted from the column with 10 ml. of anhydrous ethyl ether, and obtained as an oily concentrate by evaporation of the solvent in vacua. For the assay, the ether residue was dissolved in 4 ml. of absolute ethanol. Two milliliters of this solution was used for a blank while the other 2 ml. was used for the color reaction. This 2-ml. solution was treated with 0.5 ml. of pure ethyl cyanoacetate, and then was thoroughly mixed with 0.5 ml. of 0.2 N pot,assium hydroxide solution. A blue color slowly formed and reached its peak intensity in 6-10 min. then faded gradually on longer standing. The density reading at 625 rnp was taken exactly 10 min. after the addition of the potassium hydroxide solution. One-centimeter cells in a Beckman DU spectrophotometer equipped with a tungsten lamp were used. For the blank, the 2-ml. aliquot was treated with 0.5 ml. ethanol (in place of the ethy1 cyanoacetate) and 0.5 ml. of 0.2 N potassium hydroxide solution. The density of this solution was determined similarly after a IO-min. waiting period.
COENZYME
A calibration curve was prepared by determining the optical densities of several concentrations of known amount of pure Q1o under these conditions. The amount of Q10 present in an unknown sample was determined by evaluating its optical density on the straight-line graph. By adhering to strict standardization throughout the assay, it was found possible to detect as little as 10 pg. Qlo . Since only one-half of the concentrate was used in the color reaction, the total amount of &IO in the 24-hr. urine collection is twice that found in the aliquot. Reproducibility of Assay Procedure. Using this procedure, the reproducibility of the assay was checked by extracting and assaying several 1800ml. aliquots from a single pooled sample of normal male urine. Table I lists the results obtained from several assays of each of three different collections of urine. RECOVERY TO
OF COENZYME HUMAN URINE
Qlo ADDED
An 1800~ml. aliquot of a pooled collection of urine was diluted with an equal volume of denatured alcohol and then extracted twice with 1800-ml. portions of Skellysolve B. The SkelWysolveextract was washed with 1800ml. water, dried with 25 g. of anhydrous sodium sulfate, then concentrated to dryness in vacua. The residue was dissolved in 10 ml. of Skellysolve B, and the solution was chromatographed over a 7 mm. by 12 cm. column of acid-washed alumina using slight air pressure. The column was washed with another 10 ml. of Skellysolve B. The Q10 remained near the top of the column in a narrow yellow band, and was eluted with 10 ml. of an.hydrous ethyl ether. The colorimetric assa,y of this ether residue showed the presence of 50 pg. &lo in the 1800-ml. urine sample. The urine was then extracted twice more with Skellyaolve B. Assay as above of these pooled extracts showed no measurable amount of I&O . The residual amount of solvent in the aqueous solution was removed in vacua at, 25-30”, and a solution of 100 pg. of pure coenzyme &lo in 2 ml. alcohol was added. After standing at room temperature for 3 hr., the solution was dilut,ed with an equal volume of denatured alcohol, then extracted, processed, and assayed as above. The calorimetric assay showed the presence of 98 pg. Qlo, or a 98 % recovery of the added compound.
0.
301
XIII
TABLE
I
REPRODUCIBILITY Pooled urine collection
STUDY Qlo found in 18Wml. aliquots P&T.
A
90, 87, 90 58, 60, 55, 60 122, 126, 120,120
B C
In another experiment, two 1800-ml. aliquots from another pooled collection of urine were used. The amount of Q10present in one aliquot was determined by processing as described above. This pooled collection was found to contain 30 pg. Q10. The second 1800-ml. aliquot was then enriched by the addition of 100 pg. of pure Q10 dissolved in 2 ml. alcohol. After 3 hr., this enriched urine was extracted, processed, and assayed. This assay showed the presence of 130 fig. Q10, or a 100% recovery. In several other similar experiments, the recovery of added Q10from the urine averaged better than 95 %. STABILITY IN
Qlo
OF COENZYME HUMAN URINE
An 1800-ml. aliquot of a pooled collection of urine was extracted, processed, and assayed. This aliquot contained 50 pg. Q10. Another 1800-ml. aliquot from this collection was allowed to stand at room temperature without added preservative for 3 days. At the end of this time, the urine was cloudy and somewhat malodorous, but its pH was still 6.5. The assay showed the presence of 50 cLg.&IO. This result was verified in three repeat experiments. In one case, the pH increased from 6.5 to 7.5, but the &lo content remained unchanged. These results indicated that the Q1owas stable in urine at room temperature for at least 3 days, and that bacterial growth in the urine did not alter the quantity of &lo found. THE
COENZYME
Qlo HUMAN
CONTENT URINE
OF
NORMAL
All the assays of Q1,, levels in individual urines
reported
were
made
on 24-hr.
collec-
tions for metabolic significance. Furthermore, an entire collection was processedfor assay, because the concentration of &IO in urine is low and the lower limit of detection
302
KONIUSZY,
GALE,
of the color reaction is about 10 pg. Collections which showed less than 10 pg. are reported as “0.” The individuals involved were all fully aware of the importance of complete 24-hr. collections for this study. Consequently, creatinine excretion data were considered unnecessary. Altogether, 160 individual urine collections from 41 normal males and 22 normal females were assayed. Two pregnant women were included. All samples were processed on the day of final voiding. In many cases, several collections from the same individual were examined, particularly when an individual was found who excreted “0”-levels of &lo. The pertinent data, including ages and weights are shown in Table II. TABLE
II
Sex, Weight
Days from 1st Collection
M 160 M 164 M 139 M 155 M 52 M 148 M 200 M 168 M 168 M 195
FOLKERS
TABLE No. (Age)
0 4 9 0 56 62 0 10 21 0 6 17 0 9 0 17 30 0 9 43 0 11 16 0
142 166 115 52 86 66 60 70 60 155 105 40 40 45 46 40 52 “0”
0 78
50 76
35 130 75 35 135 30
II-Continued
SCS, Weight
Days from 1st Collection
M 180 M 178
0 21 0 11 23 0 10 16 0 10 16 0 1 2 28 0 30 32 0 5 0 29 31 0
QlO
P&T.
M 168 M 170 M 162
M 172
QIO
PA-. (3:)
AND
M 158 M 182
COENZYME Qlo CONTENT OF 24-HR. URINE COLLECTIONS No. (Age)
PAGE
M 140 M 162 M 170 M 169 M 165 M 168 M 180 M 129 M 162 v 222 M 175 M
35 50 “0” “0” 32 “0” 40 38 “0” 90 85 30 “0” “0” 61 44 46 40 20 61 “0” 36 “0” “0”
0 30 0
“0”
0 28 29 0 1 28 0 5 0 28 30 0 4 6 0 24 20 0 90 0 2 6 0
44 46 40 30 “0” 64 20 67 “0” 36 “0” 30 52 46 26 160 43 74 70 86 66 102 38
74 53
COENZYME
TABLE No. (Age)
Sex,
Weight
Q.
303
XIII
II-Continued
TABLE
Days from Collection
1st
QlO
No. Lb)
Sex, Weight
II-Concluded Days from 1st Collection
QlO Pg.
130 M 145 M 160 M 125 M 165 M 165 M 147 M 169 M 158 M 163 M 149 M 159 F 113 F 124 F 125 F 132 F 112 F 127 F 115 F 140 F 135
2 0 9 20 0 2 9 0 2 12 0 2 7 ‘0 13 0 3 12 0
68 76 64 76 110 102 56 66 64 44 96 82 110 80 39 92 94 80 36
0 3 0 14 0
62 40 42 80 92
0 1 18 0 9 30 0 6 13 0 6 20 0 7 21 0
28 38 30 24
F 1255
(ZZ)
& F 142 F 123 F 129
F 1580 F 121 F 137 F 127
“0”
20 “0” 23 58 “0” 24 “0” “0” 26 “0” “0”
0
24
0 7 21 0 7 0 1 50
“0" 46 30 30 21 41 “0” 34
F 128
0 7 27 0 9 14 0 9 10 0 36 38 0 2 0 26 27 0 6 8 0 6 0 6 17 0 18 0 6 11 0 1 18 0 11 17
52 44 108 21 37 “0” 50 18 20 37 22 56 40 26 “0” 15 18 26 14 16 32 28 16 12 18 “0” “0” 32 “0” “0” 27 40 “0” 12 “0” ‘LO”
a Pregnant. DISCUSSION
It is evident from the graphic representation of the data in Fig. 2 that the average QIO excretion by normal males was 55 pg./24 hr. The average value for females was cu. 22 pg. Figure 2 also shows that the individual averages fall into an essentially normal distribution about the average for the sex. The Q1o urinary levels of some individuals were reasonably constant over a period of weeks, i.e., Nos. 2, 3, 6, 22, and 36. However, &lo levels of other individuals varied widely
304
KONIUSZY,
JJ
GALE,
PAGE
G COENZYME
Q&21(
AND
FOLKERS
HRS. -
FIG.
2. Coenzyme &IO excretion
at different times, i.e., Nos. 7, 14, 15, and 20. It can be calculated from Table II that cu. 24 % of the males and 59% of the females excreted “0” levels of Q10 (i.e., O-10 pg.) in certain 24-hr. periods, and some such individuals, Nos. 12, 18, 25, 44, and 45 apparently excreted considerably lessthan the average value on several occasions. Precise determination of these very low Q levels cannot be made by the calorimetric method used for these determinations. A new, more sensitive analytical method is needed to evaluate such low levels. It is estimated from other data (7) that the total amount of &lo in a 70-kg. human may be about 500-1500 mg. Since &lo is so sparingly soluble in water (and urine) one would not expect urine to be the major excretory route of this compound. From the values reported in this paper,
Women
of normal individuals.
only 0.01% of the body content is normally eliminated as &IO by this route in 24 hr. The crux of this study is that 35 % of the individuals excreted less than 10 pg. Q/day on one or more occasions. One may reason that the periodic excretion of the “0” levels of this compound even by these “normal” individuals might signify that blood or tissue levels of Q10 had decreased significantly during such periods. Urine levels may be a sensitive index of such changes. Further data will be required to decide whether or not this interpretation is correct. Further studies, needed to establish whether normal and subnormal levels of body &IO are correlated with health and disease, are in progress. ACKNOWLEDGMENTS The advice of Dr. Augustus Gibson and of Dr. George Boxer, and the cooperation of Miss V. C.
COENZYME
Jelinek in the early exploratory fully acknowledged.
probes are grate-
REFERENCES 1. LINN,
B. O.,
PAGE,
A. C.,
JR.,
WONG,
E. L.,
GALE, P.H.,SHUNK,C.H., ANDFOLKERS, K., J. Am. Chem. Sot. 81, 4007 (1959). 2. AMBE,K.S., AND CRANE, F.L., FederationProc. 18, 181 (1959).
Q. XIII
305
3. FLEISCHER, S., AND LESTER, R. L., Federation Proc. 18,227 (1959). 4. MERVYN, L., ANDMORTON, R. A.,Biochem.J. 72, 106 (1959). 5. VANDENHEUVEL, F. H., AND VATCHER, D. R., Anal. &em. 28, 838 (1956). 6. SHUNK, C. H., MCPHERSON, J. F., AND FOLPERS, K., J. org. Chem., in press. 7. Unpublished.
OF
BIOCHEMISTRY
Coenzyme
AND
Q.
XIII.
87,
BIOPHYSICS
Isolation,
29&305-(1960)
Assay
and
of Coenzyme FRANK
R. KONIUSZY,
Merck,
Sharp
Human
Research
Received
Laboratories, October
Levels
Q,,
PAUL H. GALE, ATWOOD AND KARL FOLKERS
& Dohme
Urinary
Rahway,
C. PAGE,
New
JR.
Jersey
21, 1959
Pure coenzyme Qlo has been isolated from normal human male urine; a calorimetric assay for determining QrJevels in urine has been devised and applied to 160 collections representing 63 males and females. The average excretion is ea. 55 pg./24 hr. for males and 22 pg./24 hr. for females. Thirty-five per cent of the 63 individuals excreted
Coenzyme Qlo (I) of the coenzyme Q group (five members, i.e., Q6-Q10), exists widespread in animal species. The existence of coeneyme Qlo in the human body was estab0 CH,O
and subsequently, the amounts of the coenzyme excreted per day by a group of presumably normal individuals were determined. These data are basic for an interpretation of the analytical values of human urines from various disease states for possible guidance of exploratory medical studies. EXPERIMENTAL
CH,O 0
lished by the isolation of the crystalline substance from human heart tissue (1). Recent biochemical reports state that the coenzyme Q group is specific for restoration of cytochrome c reductase activity (2), and for restoration of succinoxidase activity of acetone-extracted mitochondria (3). Mervyn and Morton (4) have reported values, determined spectrophotometrically, of the coenzyme Q content of normal human kidney and 16 specimens of diseased kidney tissue. The content was similar in both cases. It appears that a study of a possible role of coenzyme Q10 in human health and disease would be benefited by data on the blood and urine levels of this substance. Urinary excretion was studied first, and our results are described in this publication. Evidence was sought on whether or not coenzyme Q10 is present in human urine;
AND
RESULTS
RESULTS ON ISOLATION OF COENZYME QIo Coenzyme Q1o was isolated in pure crystalline form from 140 gal. of pooled normal human male urine. It has not been possible to perform quantitative assays on urine itself, but it is possible to assay the material from the first solvent extractives. At this step, the 3.25 g. of extractives contained 16.3 mg. &lo . Crystalline Qlo (10.8 mg.) was obtained from these extractives in 66 % yield. The &lo from urine was identified by its melting point and spectral characterist.ics, including a nuclear magnetic resonance spectrum. It was examined by a radial paper chromatographic system which will separate Qro from its lower homologs (Q9 , etc.). Only Qlo was found in the crystalline product, although a spot with an R/ characteristic of Q9 was detected in the mother liquors from the crystallization. No evidence was ob298
COENZYME
served for the presence, in any of the materials, of additional members of the Q group, nor for metabolic degradation products which retained the substituted benzoquinone nucleus. Experimental A total of 1.40 gal. of normal male urine was collected over a lo-day period. Each day’s collection was processed on the following day. A given day’s collection (12-16 gal.) was concentrated in uucuo at 3(f35” to one-half volume. The concentrate was diluted with an equal volume of denatured ethyl alcohol (2BA). (Methanol should not be eubstituted for ethyl alcohol.) The mixture was then extracted with three portions of Skellysolve B, each equal in volume to the urine concentrate (6-8 gal.). Emulsions, which occasionally formed at this stage, were broken by the addition of a little more alcohol. The combmed Skellysolve extract was washed twice with one-third its volume of water and dried with 100 g. of anhydrous sodium sulfate; it was then concentrated in vacua to yield a thick yellowish gum. From the 140 gal. urine, 3.25 g. of yellow gum was obtained. An aliquot of this gum was assayed calorimetrically and showed the presence of 16.3 mg. coenzyme Q,o in the total residue. The 3.25 g. residue was dissolved in 100 ml. Skellysolve .B, and the solution was chromatographed over a 30 mm. X 12 cm. column of Florisil. The column was washed with 7 1. Skellysolve B, which did not elute a significant amount of Q1,, . The column was then eluted with 12 1. of 10% ethyl ether in Skellysolve B. Concentration of this eluate yielded I.2 g. of yellow gum, containing 12.8 mg. Q10 by c’olorimetric assay. The 1.2 g. gum was dissolved in 15 ml. of 99.37$ methanol which had been equilibrated with isooctane, and the solution was partitioned in a 20 mm. X 50 cm. column containing 100 g. silicic acid which had been silanized (5) by treatment with dimethyldichlorosilane. The partitioning was continued with methanol-isooctane until the visible yellow band was collected separately in approximately 50 mll. eluate. Concentration of this yellow eluate yielded 75 mg. of yellowish white crystalline material which showed 12.3 mg. &lo by assay. Countercurrent distribution of this 75-mg. sample through five plates with 50-ml. portions of 95% dimethylformamide (DMF)-isooctane, to remove some cholesterol, yielded a bright yellow residue from the isooctane layer in the first plate (DMF was the moving phase) that weighed 71.3 mg. This 71.3 mg. residue was dissolved in 30 ml. of absolute methanol. A. few drops of water were added to this solution, and the white precipitate which formed
Q. XIII
299
was removed by centrifugation. The dropwise additions of water and the centrifugings were continued until no more white precipitate formed. The final methanol concentration was approximately 96%. Concentration of the methanol solution to dryness in z)acuo yielded 17.0 mg. of yellow gummy residue, which was dissolved in 1.5 ml. of absolute ethyl alcohol. This solution was diluted with 9 ml. of absolute methanol, and by the following day, 11.9 mg. of yellow crystals had separated; m.p. 42-44”C. The first recrystallization gave crystals melting at 4G-48”; the second, m.p. 4%-49”; the third, m.p. 49-50”; the fourth, m.p. 49-50”. The yield of crystalline coenzyme Q,o was 10.8 mg.
PAPER
STRIP AND NMR DATA Qlo FROM URINE
ox
Samples of the crystalline product, coenzyme &lo from beef heart, and a mixture of the two were papergrammed radially using reversed-phase technique (1). The 32 cm. diameter circular sheet of Whatman No. 1 paper was prepared by passing it once through a 5 % solution of petroleum jelly (Vaseline) in petroleum ether and allowing it to dry in air. A 97:3 mixture of dimethylformamide (Merck, Reagent) and water was the mobile phase. Zones appeared as dark areas when the air-dried papergram was held over an ultraviolet lamp. The crystalline Q from urine, crystalline coenzyme Q10 from beef heart, and a mixture of the two all moved with identical Rr’s in this system, which is readily capable of distinguishing Qlo from other members of the group. The nuclear magnetic resonance spectra of the two crystalline materials were identical.
QUANTITATIVE DETERMINATION IN URINE SAMPLES
OF Qlo
It appeared that the modified Craven’s test, which has been examined (6) with a considerable array of synthetic model quinones and members of the Q group, could be developed into a calorimetric assay for Qlo in urines. The method involves the reaction of &lo and ethyl cyanoacetate in the presence of potassium hydroxide. The blue color which forms is measured after 10 min. A standard curve is shown in Fig. 1. Zero levels (“O”levels) or O-10 pg. signify that there was no
300
KONIUSZY,
GALE,
color; lo-25-pg. levels are estimates based on very weak color reactions; higher levels of the colored Q10 reaction product are measured with more precision. In replicate determinations of the same sample, a standard deviation of f8 pg. was found for any of the levels studied in the lo-lOO+g. range. Greater variation (~15 %) was found in the optical density observed with a given level of Q1o standard on different days. However, all of the levels in the standard in a single assay were altered proportionately. This reaction gives reproducible results with partially purified concentrates from urine samples as well as with crystalline &lo . The established procedure consists of direct extraction of the urine sample and further purification of the extractives by alumina chromatography. Florisil, Decalso, acid-washed alumina, and silicic acid all gave recoveries of over 80%; but the best recoveries (95-100%) were obtained with acid-washed alumina columns. Q10 is decomposed if it is allowed to remain longer than 24 hr. on alumina columns. Since the chromatographic step is completed in about 1 hr., significant losses are avoided. Assay of several aliquots of several collections of pooled urine showed that the varia-
FIG. mination
1. Standard curve for of Coenzyme &IO .
calorimetric
deter-
PAGE
AND
FOLKERS
tion in the assay of a single collection was less than &5%. When pure coenzyme Qio was added to several assayed urine collections, the recoveries of the added QN were 95-100 %. Experimental Procedure. For analysis of the total amount of coenzyme &lo eliminated in human urine over a 24-hr. period, the following procedure was found to be the most convenient and reproducible. The individual urine samples were collected without added preservative. If the determination could be carried out within 1 or 2 days after the final voiding, no detectable loss of &lo was observed. The volume and the pH of the urine were noted. The urine was diluted with an equal volume of denatured alcohol, and this mixture was then shaken vigorously with a volume of Skellysolve B equal to the original volume of urine. The alcoholdiluted urine was separated and discarded. The Skellysolve layer was washed by shaking it with an equal volume of distilled water, separated, then dried by shaking it with 25 g. of anhydrous sodium sulfate. The Skellysolve extract was carefully decanted into a concentration flask without. filtration, since filter paper tends to adsorb Q10 . After the extract was concentrated to dryness in vacua, the residue was dissolved in 10 ml. of Skellysolve B. This solution was chromatographed over a 7 mm. diameter by 12 cm. column of acid-washed alumina. Solvent flow was increased by the application of 2-3 lb./sq. in. air pressure to the top of the column. The chromatogram was washed with 10 ml. of Skellysolve B, and this washing was discarded. The coenzyme Q was eluted from the column with 10 ml. of anhydrous ethyl ether, and obtained as an oily concentrate by evaporation of the solvent in vacua. For the assay, the ether residue was dissolved in 4 ml. of absolute ethanol. Two milliliters of this solution was used for a blank while the other 2 ml. was used for the color reaction. This 2-ml. solution was treated with 0.5 ml. of pure ethyl cyanoacetate, and then was thoroughly mixed with 0.5 ml. of 0.2 N pot,assium hydroxide solution. A blue color slowly formed and reached its peak intensity in 6-10 min. then faded gradually on longer standing. The density reading at 625 rnp was taken exactly 10 min. after the addition of the potassium hydroxide solution. One-centimeter cells in a Beckman DU spectrophotometer equipped with a tungsten lamp were used. For the blank, the 2-ml. aliquot was treated with 0.5 ml. ethanol (in place of the ethy1 cyanoacetate) and 0.5 ml. of 0.2 N potassium hydroxide solution. The density of this solution was determined similarly after a IO-min. waiting period.
COENZYME
A calibration curve was prepared by determining the optical densities of several concentrations of known amount of pure Q1o under these conditions. The amount of Q10 present in an unknown sample was determined by evaluating its optical density on the straight-line graph. By adhering to strict standardization throughout the assay, it was found possible to detect as little as 10 pg. Qlo . Since only one-half of the concentrate was used in the color reaction, the total amount of &IO in the 24-hr. urine collection is twice that found in the aliquot. Reproducibility of Assay Procedure. Using this procedure, the reproducibility of the assay was checked by extracting and assaying several 1800ml. aliquots from a single pooled sample of normal male urine. Table I lists the results obtained from several assays of each of three different collections of urine. RECOVERY TO
OF COENZYME HUMAN URINE
Qlo ADDED
An 1800~ml. aliquot of a pooled collection of urine was diluted with an equal volume of denatured alcohol and then extracted twice with 1800-ml. portions of Skellysolve B. The SkelWysolveextract was washed with 1800ml. water, dried with 25 g. of anhydrous sodium sulfate, then concentrated to dryness in vacua. The residue was dissolved in 10 ml. of Skellysolve B, and the solution was chromatographed over a 7 mm. by 12 cm. column of acid-washed alumina using slight air pressure. The column was washed with another 10 ml. of Skellysolve B. The Q10 remained near the top of the column in a narrow yellow band, and was eluted with 10 ml. of an.hydrous ethyl ether. The colorimetric assa,y of this ether residue showed the presence of 50 pg. &lo in the 1800-ml. urine sample. The urine was then extracted twice more with Skellyaolve B. Assay as above of these pooled extracts showed no measurable amount of I&O . The residual amount of solvent in the aqueous solution was removed in vacua at, 25-30”, and a solution of 100 pg. of pure coenzyme &lo in 2 ml. alcohol was added. After standing at room temperature for 3 hr., the solution was dilut,ed with an equal volume of denatured alcohol, then extracted, processed, and assayed as above. The calorimetric assay showed the presence of 98 pg. Qlo, or a 98 % recovery of the added compound.
0.
301
XIII
TABLE
I
REPRODUCIBILITY Pooled urine collection
STUDY Qlo found in 18Wml. aliquots P&T.
A
90, 87, 90 58, 60, 55, 60 122, 126, 120,120
B C
In another experiment, two 1800-ml. aliquots from another pooled collection of urine were used. The amount of Q10present in one aliquot was determined by processing as described above. This pooled collection was found to contain 30 pg. Q10. The second 1800-ml. aliquot was then enriched by the addition of 100 pg. of pure Q10 dissolved in 2 ml. alcohol. After 3 hr., this enriched urine was extracted, processed, and assayed. This assay showed the presence of 130 fig. Q10, or a 100% recovery. In several other similar experiments, the recovery of added Q10from the urine averaged better than 95 %. STABILITY IN
Qlo
OF COENZYME HUMAN URINE
An 1800-ml. aliquot of a pooled collection of urine was extracted, processed, and assayed. This aliquot contained 50 pg. Q10. Another 1800-ml. aliquot from this collection was allowed to stand at room temperature without added preservative for 3 days. At the end of this time, the urine was cloudy and somewhat malodorous, but its pH was still 6.5. The assay showed the presence of 50 cLg.&IO. This result was verified in three repeat experiments. In one case, the pH increased from 6.5 to 7.5, but the &lo content remained unchanged. These results indicated that the Q1owas stable in urine at room temperature for at least 3 days, and that bacterial growth in the urine did not alter the quantity of &lo found. THE
COENZYME
Qlo HUMAN
CONTENT URINE
OF
NORMAL
All the assays of Q1,, levels in individual urines
reported
were
made
on 24-hr.
collec-
tions for metabolic significance. Furthermore, an entire collection was processedfor assay, because the concentration of &IO in urine is low and the lower limit of detection
302
KONIUSZY,
GALE,
of the color reaction is about 10 pg. Collections which showed less than 10 pg. are reported as “0.” The individuals involved were all fully aware of the importance of complete 24-hr. collections for this study. Consequently, creatinine excretion data were considered unnecessary. Altogether, 160 individual urine collections from 41 normal males and 22 normal females were assayed. Two pregnant women were included. All samples were processed on the day of final voiding. In many cases, several collections from the same individual were examined, particularly when an individual was found who excreted “0”-levels of &lo. The pertinent data, including ages and weights are shown in Table II. TABLE
II
Sex, Weight
Days from 1st Collection
M 160 M 164 M 139 M 155 M 52 M 148 M 200 M 168 M 168 M 195
FOLKERS
TABLE No. (Age)
0 4 9 0 56 62 0 10 21 0 6 17 0 9 0 17 30 0 9 43 0 11 16 0
142 166 115 52 86 66 60 70 60 155 105 40 40 45 46 40 52 “0”
0 78
50 76
35 130 75 35 135 30
II-Continued
SCS, Weight
Days from 1st Collection
M 180 M 178
0 21 0 11 23 0 10 16 0 10 16 0 1 2 28 0 30 32 0 5 0 29 31 0
QlO
P&T.
M 168 M 170 M 162
M 172
QIO
PA-. (3:)
AND
M 158 M 182
COENZYME Qlo CONTENT OF 24-HR. URINE COLLECTIONS No. (Age)
PAGE
M 140 M 162 M 170 M 169 M 165 M 168 M 180 M 129 M 162 v 222 M 175 M
35 50 “0” “0” 32 “0” 40 38 “0” 90 85 30 “0” “0” 61 44 46 40 20 61 “0” 36 “0” “0”
0 30 0
“0”
0 28 29 0 1 28 0 5 0 28 30 0 4 6 0 24 20 0 90 0 2 6 0
44 46 40 30 “0” 64 20 67 “0” 36 “0” 30 52 46 26 160 43 74 70 86 66 102 38
74 53
COENZYME
TABLE No. (Age)
Sex,
Weight
Q.
303
XIII
II-Continued
TABLE
Days from Collection
1st
QlO
No. Lb)
Sex, Weight
II-Concluded Days from 1st Collection
QlO Pg.
130 M 145 M 160 M 125 M 165 M 165 M 147 M 169 M 158 M 163 M 149 M 159 F 113 F 124 F 125 F 132 F 112 F 127 F 115 F 140 F 135
2 0 9 20 0 2 9 0 2 12 0 2 7 ‘0 13 0 3 12 0
68 76 64 76 110 102 56 66 64 44 96 82 110 80 39 92 94 80 36
0 3 0 14 0
62 40 42 80 92
0 1 18 0 9 30 0 6 13 0 6 20 0 7 21 0
28 38 30 24
F 1255
(ZZ)
& F 142 F 123 F 129
F 1580 F 121 F 137 F 127
“0”
20 “0” 23 58 “0” 24 “0” “0” 26 “0” “0”
0
24
0 7 21 0 7 0 1 50
“0" 46 30 30 21 41 “0” 34
F 128
0 7 27 0 9 14 0 9 10 0 36 38 0 2 0 26 27 0 6 8 0 6 0 6 17 0 18 0 6 11 0 1 18 0 11 17
52 44 108 21 37 “0” 50 18 20 37 22 56 40 26 “0” 15 18 26 14 16 32 28 16 12 18 “0” “0” 32 “0” “0” 27 40 “0” 12 “0” ‘LO”
a Pregnant. DISCUSSION
It is evident from the graphic representation of the data in Fig. 2 that the average QIO excretion by normal males was 55 pg./24 hr. The average value for females was cu. 22 pg. Figure 2 also shows that the individual averages fall into an essentially normal distribution about the average for the sex. The Q1o urinary levels of some individuals were reasonably constant over a period of weeks, i.e., Nos. 2, 3, 6, 22, and 36. However, &lo levels of other individuals varied widely
304
KONIUSZY,
JJ
GALE,
PAGE
G COENZYME
Q&21(
AND
FOLKERS
HRS. -
FIG.
2. Coenzyme &IO excretion
at different times, i.e., Nos. 7, 14, 15, and 20. It can be calculated from Table II that cu. 24 % of the males and 59% of the females excreted “0” levels of Q10 (i.e., O-10 pg.) in certain 24-hr. periods, and some such individuals, Nos. 12, 18, 25, 44, and 45 apparently excreted considerably lessthan the average value on several occasions. Precise determination of these very low Q levels cannot be made by the calorimetric method used for these determinations. A new, more sensitive analytical method is needed to evaluate such low levels. It is estimated from other data (7) that the total amount of &lo in a 70-kg. human may be about 500-1500 mg. Since &lo is so sparingly soluble in water (and urine) one would not expect urine to be the major excretory route of this compound. From the values reported in this paper,
Women
of normal individuals.
only 0.01% of the body content is normally eliminated as &IO by this route in 24 hr. The crux of this study is that 35 % of the individuals excreted less than 10 pg. Q/day on one or more occasions. One may reason that the periodic excretion of the “0” levels of this compound even by these “normal” individuals might signify that blood or tissue levels of Q10 had decreased significantly during such periods. Urine levels may be a sensitive index of such changes. Further data will be required to decide whether or not this interpretation is correct. Further studies, needed to establish whether normal and subnormal levels of body &IO are correlated with health and disease, are in progress. ACKNOWLEDGMENTS The advice of Dr. Augustus Gibson and of Dr. George Boxer, and the cooperation of Miss V. C.
COENZYME
Jelinek in the early exploratory fully acknowledged.
probes are grate-
REFERENCES 1. LINN,
B. O.,
PAGE,
A. C.,
JR.,
WONG,
E. L.,
GALE, P.H.,SHUNK,C.H., ANDFOLKERS, K., J. Am. Chem. Sot. 81, 4007 (1959). 2. AMBE,K.S., AND CRANE, F.L., FederationProc. 18, 181 (1959).
Q. XIII
305
3. FLEISCHER, S., AND LESTER, R. L., Federation Proc. 18,227 (1959). 4. MERVYN, L., ANDMORTON, R. A.,Biochem.J. 72, 106 (1959). 5. VANDENHEUVEL, F. H., AND VATCHER, D. R., Anal. &em. 28, 838 (1956). 6. SHUNK, C. H., MCPHERSON, J. F., AND FOLPERS, K., J. org. Chem., in press. 7. Unpublished.