Life Sciences, Vol. 47, pp. 2135-2141 Printed in the U.S.A.
Pergamon Press
EXTRACTIONS OF PYRROLOQUINOLINE QUINONE FROM CRUDE BIOLOGICAL SAMPLES
O. Suzuki , T. Kumazawa , H. Seno*, T. Urakami** and T. Matsumoto***
Department of Legal Medicine, Hamamatsu U n i v e r s i t y School of Medicine, Hamamatsu
431-31;
Mitsubishi Gas Chemical Company, Inc.,
Tokyo 105; and ***First Department of Surgery, Nagoya University School of Medicine, Nagoya 466, Japan (Received in final form October I, 1990) Summary The best conditions for extractions of free pyrroloquinoline quinone (PQQ) from crude biological samples were investigated with various organic solvents and Sep-Pak C18 cartridges. PQQ was measured with use of its native fluorescence in aqueous solution. PQQ was well extracted into n-butanol under acid conditions, and addition of NaC1 did not improve the solvent extraction. PQQ, which had been extracted into n--butanol, could be re-extracted into an aqueous phase by addition of either ~-heptane or pyridine, or combination of them. PQQ, which had been adsorbed to Sep-Pak C18 cartridges, could be eluted with a mixture of pyridine and water with very excellent recovery. The recovery of I ~g PQQ, which had been added to I g human liver, brain and I ml plasma and had undergone the n_-butanol and the Sep-Pak extractions, was 50, 75 and 105 %, respectively. From the blank fluorescence, endogenous levels of free PQQ in human liver, brain and plasma were found not greater than 0.41, 0.08 and 0.13 ~ug/g or ml, respectively, if present.
Pyrroloquinoline quinone (PQQ) was identified as a novel co-factor in various dehydrogenases purified from bacteria in early studies (1,2). Recently, a few research groups have claimed that covalently bound PQQ is located in various mammalian enzymes, such as amine oxidases (3-6) and lysyl oxidase (7,8), suggesting ubiquitous presence of PQQ in mammalian tissues. More recently, it has been reported that PQQ is nutritionally important as a vitamin or growth factor ( 9 ) . However, no one has succeeded in detecting free PQQ in mammalian tissues. This seems partly because no extraction methods have been established for PQQ. In this report, we present simple methods for extracting PQQ from crude tissues, which are applicable to various PQQ studies.
Materials and Methods MATERIALS. PQQ was obtained from Mitsubishi Gas Chemical Company, Inc. (Niigata, Japan); Sep-Pak C18 cartridges from Waters Associates (Milford, MA). Other common chemicals used were of the highest purity commercially 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
2136
PQQ Extraction From Crude Samples
Vol. 47, No. 23, 1990
available. Human liver and brain were obtained at forensic plasma from Hamamatsu Red Cross Center (Hamamatsu, Japan).
autopsies;
human
FLUOROMETRIC MEASUREMENTS OF PQQ. PQQ in 0.1 N HCI solution was quantitated with its fluorescence (10), with excitation at 375 nm and emission at 465 nm (uncorrected), on a Hitachi 650-IOS fluorescence spectrophotometer. ORGANIC SOLVENT EXTRACTION. Three milliliters of each organic solvent listed in Table I and 3 ml 0 . 1 N HCI solution were shaken vigorously in the presence of 1 jug PQQ, and c e n t r i f u g e d at 500 g for 5 min. The aqueous phase was subjected to the fluorometric measurements. The aqueous phase after shaking with an organic solvent in the absence of PQQ served as a blank test. The standard sample was made by adding l ~ g PQQ, which had been dissolved in I ~ i water, to the final aqueous phase of the blank test. The same line of experiments was also carried out in the presence of 0.8 g NaCI. From the d e c r e a s e in PQQ in the aqueous phase after organic solvent extraction, partition coefficients of it were calculated. EXTRACTION OF PQQ FROM n-BUTANOL BACK INTO AN AQUEOUS PHASE. To test reextraction of PQQ, which had been extracted into ~-butanol, back into an aqueous phase, 3 ml n-butanol saturated with 0 . 1 N HCI was shaken with 3 ml water and I ~ g PQQ in the presence of NaCI, n - h e p t a n e or pyridine, or combination of them as shown in Table II. The mixtures were centrifuged at 500 g for 5 min, and each aqueous phase was subjected to the fluorometric measurements. In these experiments, each blank test was carefully prepared by shaking the mixtures without addition of PQQ. As a standard sample, I ~ g PQQ was added to the final aqueous phase of each blank test. ISOLATION WITH SEP-PAK C18 CARTRIDGES. For pretreatment of a Sep-Pak C cartridge, 10 ml 5 % p y r i d i n e in water, 10 ml a c e t o n i t r i l e and 10 m~ distilled water were passed through it. Recoveries of PQQ during its elution from S e p - P a k C18 cartridges were tested with 3 d i f f e r e n t eluents. One microgram of PQQ dissolved in 10 ml O . 1 N HCI was loaded on the cartridges. After washing the cartridges with 20 ml 0.001 N HCI, the adsorbed PQQ was eluted with 5 % pyridine (in water), acetonitrile or methanol, 3 ml of each. They were evaporated to dryness in vacuo and the residues dissolved in 3 ml O . 1 N HCI for the fluorometric measurements. Blank and standard tests were also prepared for each elution solvent. E X T R A C T I O N OF PQQ F R O M CRUDE B I O L O G I C A L SAMPLES. To 1 g tissue or I ml plasma including I ~ g PQQ, were added 4 ml I N HCI, 40 ~ i mercaptoethanel and 0.1 ml 10 % p o t a s s i u m f e r r i c y a n i d e . The tissue was minced with surgery scissors. After addition of 10 ml n-butanol to the above mixture, it was homogenized with a Polytoron homogenizer for 5 min being cooled with ice and c e n t r i f u g e d at 500 g for 5 min. After the organic layer was c a r e f u l l y transferred to another centrifuge tube, I ml distilled water, 1 ml pyridine, O.1 g NaCI and 20 ml ~ - h e p t a n e were added to it, shaken v i g o r o u s l y and c e n t r i f u g e d as above. The aqueous layer (lower phase) was c a r e f u l l y transferred to a 20 ml-test tube and evaporated in vacuo only for 15 min to remove the residual organic solvents from the aqueous solution completely. It was then mixed with 10 ml 0.1 N HCI solution and passed through the pretreated Sep-Pak C18 cartridge at a flow rate not greater than 5 ml/min. The c a r t r i d g e was washed with 20 ml 0.001 N HCI, and finally 3 ml 5 % pyridine in water was passed t h r o u g h it to elute PQQ. The eluates were evaporated to dryness in vacuo and their residues dissolved in 3 ml 0.1 N HCI solution for their fluorometric measurements. An extract, without addition of PQQ at the initial step, was taken as a blank test for each tissue. As a standard, 1 ~g PQQ was added to the final residue of each blank sample.
Vol. 47, No. 23, 1990
PQQ Extraction From Crude Samples
2137
TABLE I Partition coefficients of PQQ with various organic the presence and absence of NaCl. Solvent
- NaCI
n-Hexane
solvents in
+ NaCI a
0.06
0.04
Pergamon Press
EXTRACTIONS OF PYRROLOQUINOLINE QUINONE FROM CRUDE BIOLOGICAL SAMPLES
O. Suzuki , T. Kumazawa , H. Seno*, T. Urakami** and T. Matsumoto***
Department of Legal Medicine, Hamamatsu U n i v e r s i t y School of Medicine, Hamamatsu
431-31;
Mitsubishi Gas Chemical Company, Inc.,
Tokyo 105; and ***First Department of Surgery, Nagoya University School of Medicine, Nagoya 466, Japan (Received in final form October I, 1990) Summary The best conditions for extractions of free pyrroloquinoline quinone (PQQ) from crude biological samples were investigated with various organic solvents and Sep-Pak C18 cartridges. PQQ was measured with use of its native fluorescence in aqueous solution. PQQ was well extracted into n-butanol under acid conditions, and addition of NaC1 did not improve the solvent extraction. PQQ, which had been extracted into n--butanol, could be re-extracted into an aqueous phase by addition of either ~-heptane or pyridine, or combination of them. PQQ, which had been adsorbed to Sep-Pak C18 cartridges, could be eluted with a mixture of pyridine and water with very excellent recovery. The recovery of I ~g PQQ, which had been added to I g human liver, brain and I ml plasma and had undergone the n_-butanol and the Sep-Pak extractions, was 50, 75 and 105 %, respectively. From the blank fluorescence, endogenous levels of free PQQ in human liver, brain and plasma were found not greater than 0.41, 0.08 and 0.13 ~ug/g or ml, respectively, if present.
Pyrroloquinoline quinone (PQQ) was identified as a novel co-factor in various dehydrogenases purified from bacteria in early studies (1,2). Recently, a few research groups have claimed that covalently bound PQQ is located in various mammalian enzymes, such as amine oxidases (3-6) and lysyl oxidase (7,8), suggesting ubiquitous presence of PQQ in mammalian tissues. More recently, it has been reported that PQQ is nutritionally important as a vitamin or growth factor ( 9 ) . However, no one has succeeded in detecting free PQQ in mammalian tissues. This seems partly because no extraction methods have been established for PQQ. In this report, we present simple methods for extracting PQQ from crude tissues, which are applicable to various PQQ studies.
Materials and Methods MATERIALS. PQQ was obtained from Mitsubishi Gas Chemical Company, Inc. (Niigata, Japan); Sep-Pak C18 cartridges from Waters Associates (Milford, MA). Other common chemicals used were of the highest purity commercially 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
2136
PQQ Extraction From Crude Samples
Vol. 47, No. 23, 1990
available. Human liver and brain were obtained at forensic plasma from Hamamatsu Red Cross Center (Hamamatsu, Japan).
autopsies;
human
FLUOROMETRIC MEASUREMENTS OF PQQ. PQQ in 0.1 N HCI solution was quantitated with its fluorescence (10), with excitation at 375 nm and emission at 465 nm (uncorrected), on a Hitachi 650-IOS fluorescence spectrophotometer. ORGANIC SOLVENT EXTRACTION. Three milliliters of each organic solvent listed in Table I and 3 ml 0 . 1 N HCI solution were shaken vigorously in the presence of 1 jug PQQ, and c e n t r i f u g e d at 500 g for 5 min. The aqueous phase was subjected to the fluorometric measurements. The aqueous phase after shaking with an organic solvent in the absence of PQQ served as a blank test. The standard sample was made by adding l ~ g PQQ, which had been dissolved in I ~ i water, to the final aqueous phase of the blank test. The same line of experiments was also carried out in the presence of 0.8 g NaCI. From the d e c r e a s e in PQQ in the aqueous phase after organic solvent extraction, partition coefficients of it were calculated. EXTRACTION OF PQQ FROM n-BUTANOL BACK INTO AN AQUEOUS PHASE. To test reextraction of PQQ, which had been extracted into ~-butanol, back into an aqueous phase, 3 ml n-butanol saturated with 0 . 1 N HCI was shaken with 3 ml water and I ~ g PQQ in the presence of NaCI, n - h e p t a n e or pyridine, or combination of them as shown in Table II. The mixtures were centrifuged at 500 g for 5 min, and each aqueous phase was subjected to the fluorometric measurements. In these experiments, each blank test was carefully prepared by shaking the mixtures without addition of PQQ. As a standard sample, I ~ g PQQ was added to the final aqueous phase of each blank test. ISOLATION WITH SEP-PAK C18 CARTRIDGES. For pretreatment of a Sep-Pak C cartridge, 10 ml 5 % p y r i d i n e in water, 10 ml a c e t o n i t r i l e and 10 m~ distilled water were passed through it. Recoveries of PQQ during its elution from S e p - P a k C18 cartridges were tested with 3 d i f f e r e n t eluents. One microgram of PQQ dissolved in 10 ml O . 1 N HCI was loaded on the cartridges. After washing the cartridges with 20 ml 0.001 N HCI, the adsorbed PQQ was eluted with 5 % pyridine (in water), acetonitrile or methanol, 3 ml of each. They were evaporated to dryness in vacuo and the residues dissolved in 3 ml O . 1 N HCI for the fluorometric measurements. Blank and standard tests were also prepared for each elution solvent. E X T R A C T I O N OF PQQ F R O M CRUDE B I O L O G I C A L SAMPLES. To 1 g tissue or I ml plasma including I ~ g PQQ, were added 4 ml I N HCI, 40 ~ i mercaptoethanel and 0.1 ml 10 % p o t a s s i u m f e r r i c y a n i d e . The tissue was minced with surgery scissors. After addition of 10 ml n-butanol to the above mixture, it was homogenized with a Polytoron homogenizer for 5 min being cooled with ice and c e n t r i f u g e d at 500 g for 5 min. After the organic layer was c a r e f u l l y transferred to another centrifuge tube, I ml distilled water, 1 ml pyridine, O.1 g NaCI and 20 ml ~ - h e p t a n e were added to it, shaken v i g o r o u s l y and c e n t r i f u g e d as above. The aqueous layer (lower phase) was c a r e f u l l y transferred to a 20 ml-test tube and evaporated in vacuo only for 15 min to remove the residual organic solvents from the aqueous solution completely. It was then mixed with 10 ml 0.1 N HCI solution and passed through the pretreated Sep-Pak C18 cartridge at a flow rate not greater than 5 ml/min. The c a r t r i d g e was washed with 20 ml 0.001 N HCI, and finally 3 ml 5 % pyridine in water was passed t h r o u g h it to elute PQQ. The eluates were evaporated to dryness in vacuo and their residues dissolved in 3 ml 0.1 N HCI solution for their fluorometric measurements. An extract, without addition of PQQ at the initial step, was taken as a blank test for each tissue. As a standard, 1 ~g PQQ was added to the final residue of each blank sample.
Vol. 47, No. 23, 1990
PQQ Extraction From Crude Samples
2137
TABLE I Partition coefficients of PQQ with various organic the presence and absence of NaCl. Solvent
- NaCI
n-Hexane
solvents in
+ NaCI a
0.06
0.04
