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PET imaging of 11C-labeled coenzyme Q10: Comparison of biodistribution between [11C]ubiquinol-10 and [11C]ubiquinone-10
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
PET imaging of 11C-labeled coenzyme Q10: Comparison of biodistribution between [11C]ubiquinol-10 and [11C]ubiquinone-10 Kyosuke Watanabe a, b, c, d, Satoshi Nozaki a, b, Miki Goto a, b, Ken-ichi Kaneko a, b, Emi Hayashinaka a, b, Satsuki Irie a, b, Akira Nishiyama e, Kazuaki Kasai f, Kenji Fujii e, Yasuhiro Wada a, b, Kei Mizuno a, b, c, d, Kenji Mizuseki d, Hisashi Doi a, b, Yasuyoshi Watanabe a, b, c, * a
RIKEN Center for Life Science Technologies, Japan RIKEN Center for Biosystems Dynamics Research, Japan c RIKEN Compass to Healthy Life Research Complex Program, Kobe, Hyogo, Japan d Osaka City University Graduate School of Medicine, Osaka, Japan e KANEKA Corporation, Osaka, Japan f Takasago Analysis Center, Kaneka Techno Research Corporation, Takasago, Hyogo, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 March 2019 Accepted 13 March 2019 Available online xxx
Coenzyme Q10 (CoQ10) plays a key role not only as an essential electron carrier in the mitochondrial electron transport chain, but also as an antioxidant to protect cells from oxidative stress. CoQ10 supplementation is expected to be effective for a variety of diseases. The predominant forms of CoQ10 are the ubiquinol-10 (reduced form) and ubiquinone-10 (oxidized form). Both forms of CoQ10 supplements are commercially available, however, their kinetic difference is still unclear. In order to conduct in vivo analysis of the kinetics of ubiquinol-10 and ubiquinone-10, we succeeded in synthesizing 11C-labeled ubiquinol-10 ([11C]UQL) and ubiquinone-10 ([11C]UQN), respectively. In the present study, we aimed to investigate the kinetics of [11C]UQL and [11C]UQN, both of which were administered via the tail vein of 8week-old male Sprague-Dawley rats. Whole-body positron emission tomography (PET) imaging was performed to follow the time course of accumulation in the liver, spleen, brain, and other organs. Then, at the two typical time points at 20 or 90 min after injection, we conducted the biodistribution study. Various organs/tissues and blood were collected, weighed and counted with a gamma counter. Percent injected dose per gram of tissue (%ID/g) was calculated as the indicator of the accumulation of each compound. As the results, at both time points, %ID/g of [11C]UQL in the cerebrum, cerebellum, white adipose tissue, muscle, kidney, and testis were higher (P < 0.05) than that of [11C]UQN: at 90-min time point, %ID/g of [11C]UQL in the brown adipose tissue was higher (P < 0.05) than that of [11C]UQN: on the contrary, %ID/g of [11C]UQL in the spleen was lower (P < 0.05) than that of [11C]UQN at 90 min. In a separate study of the metabolite analysis in the plasma, UQL injected into the tail vein of rats was almost unchanged during the PET scanning time, but UQN was gradually converted to the reduced form UQL. Therefore, the uptake values of UQL into the tissues and organs were rather accurate but those of UQN might be the sum of UQN uptake and partly converted UQL uptake. These studies suggested that the accumulation level of administered CoQ10 differs depending on its redox state, and that CoQ10 redox state could be crucial for optimization of the effective supplementation. © 2019 Elsevier Inc. All rights reserved.
Keywords: Coenzyme Q10 Ubiquinol Ubiquinone PET Biodistribution Metabolism
1. Introduction
* Corresponding author. RIKEN Compass to Healthy Life Research Complex Program, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan. E-mail addresses: [email protected] (K. Watanabe), [email protected] (Y. Watanabe).
Coenzyme Q10 (CoQ10) plays a key role not only as an essential electron carrier in the mitochondrial electron transport chain, but also as an anti-oxidant to protect cells from oxidative stress [1,2]. CoQ10 supplementation is expected to be effective for a variety of
https://doi.org/10.1016/j.bbrc.2019.03.073 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: K. Watanabe et al., PET imaging of 11C-labeled coenzyme Q10: Comparison of biodistribution between [11C]ubiquinol10 and [11C]ubiquinone-10, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.03.073
2
K. Watanabe et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
diseases such as neurodegenerative disease [3e6], neuropsychiatric disorders [7], myalgic encephalomyelitis/chronic fatigue syndrome [8], cardiovascular insults [9], and male infertility [10]. The predominant forms of CoQ10 are the ubiquinol-10 (reduced form) and ubiquinone-10 (oxidized form). Both forms of CoQ10 supplements are commercially available, however, their kinetic difference is still unclear. In order to conduct in vivo analysis of the kinetics of ubiquinol-10 and ubiquinone-10 not only in animals but also in human, we succeeded in synthesizing 11C-labeled ubiquinol-10 ([11C]UQL) and ubiquinone-10 ([11C]UQN), respectively [11]. In the present study, we aimed to investigate the difference of kinetics of [11C]UQL and [11C]UQN injected intravenously.
2. Materials and methods 2.1. Experimental animals Male Sprague-Dawley rats (8e9 weeks old) were sourced from CLEA Japan, Inc. (Tokyo, Japan). They were housed in a light- and temperature-controlled environment with unlimited access to food and water available. All experimental protocols were approved by the Ethics Committee on Animal Care and Use of RIKEN and were performed in accordance with the National Institute of Health guide for the care and use of Laboratory animals.
2.5. Plasma metabolite analysis The reduced form UQL is easily oxidized onto the thin-layer chromatography, possibly by activated silica. Therefore, in this case, we could not conduct the radio-metabolite analysis with rapid analytical methods routinely used in most of PET study. We chose the HPLC methods with longer time consumption, so that in place of labeled compounds we injected the unlabeled UQL or UQN into the rats and took plasma samples (n ¼ 3 or 4 for each condition) with time for LC-MS/MS analyses [12]. UQL and UQN concentration in the plasma was measured using LC-MS/MS equipped with a tandem mass spectrometer at Kaneka Techno Research Corporation. Briefly, 0.7 mL of extraction solvent was added to 0.1 mL of plasma and mixed. After centrifugation, the supernatant was filtered through a membrane filter and used as a sample for LC-MS/ MS. Measurements were made using a QTRAP® 5500 LC-MS/MS system (AB Sciex, Framingham, Mass., USA). 2.6. Statistical analysis Two-way analysis of variance followed by a post-hoc multiple comparisons test (Bonferroni correction) was performed using GraphPad Prism 6 (GraphPad Software, CA, USA). P values of less than 0.05 were considered statistically significant. 3. Results
2.2. Synthesis of
11
C-labeled ubiquinone-10 and ubiquinol-10
Carbon-11-labeled ubiquinonol-10 and ubiquinone-10 were synthesized by the method recently developed and reported by our group [11]. The specification of these labeled compounds provided for the present study were as follows: The molar activity and injected mass of [11C]UQL and [11C]UQN at the time of intravenous administration to rats were 66.7 ± 6.8 GBq/mmol (1.08 ± 0.11 nmol) and 55.2 ± 10.3 GBq/mmol (1.31 ± 0.18 nmol) (mean ± SD), respectively. The radiochemical purity and chemical purity of [11C]UQL were 97.5 ± 1.7% and 77.8 ± 7.8%, and those of [11C]UQN were 96.8 ± 3.2% and 96.7 ± 2.7%, respectively. 2.3. Positron emission tomography (PET) imaging The rats were anesthetized with 1.5% isoflurane and the cannula to each rat was inserted into their tail vein. They were placed on the PET scanner (microPET Focus 220; Siemens, Knoxville, TN, USA) and injected the radiolabeled compound (ca. 75 MBq per animal) via the tail vein. Emission data were acquired for 90 min using the continuous bed motion acquisition method. Following acquisition, the emission data were sorted into a 2D data set using Fourierrebinning algorithm and reconstructed with a maximum likelihood expectation maximization algorithm.
2.4. Biodistribution studies At 20 min and 90 min after the administration of [11C]UQL or [11C]UQN via a tail vein, the rats were sacrificed, and tissues of interest, blood and urine were collected and weighed (n ¼ 6 for each condition). Their radioactivity was measured using the scintillation counter (Wallac Wizard 1480/2480; PerkinElmer, Waltham, MA, USA). Tissues collected were cerebrum, cerebellum, heart, lung, liver, spleen, kidney, pancreas, quadriceps femoris muscle, brown adipose tissue, white adipose tissue, and testis. Percent injected dose per gram of tissue (%ID/g) was calculated as the indicator of the accumulation of the injected compound.
3.1. PET imaging Integrated whole-body maximum intensity projection images at the corresponding time periods after injection are successfully visualized (Fig. 1). Accumulation of [11C]UQL and [11C]UQN were mainly observed in the liver, lung, and spleen. We could also see the difference in the heart and aortas and head regions including the brain, somewhat higher uptake was seen by [11C]UQL. 3.2. Biodistribution studies As shown in Fig. 2, at both time points (20 and 90 min after injection), %ID/g of [11C]UQL in the cerebrum, cerebellum, white adipose tissue, muscle, kidney, and testis were higher (P < 0.05) than that of [11C]UQN, whereas that of [11C]UQL in the spleen at 90 min was lower (P < 0.05) than that of [11C]UQN. %ID/g of [11C] UQL in the brown adipose tissue was higher (P < 0.05) than that of [11C]UQN at 90 min after injection. Blood concentration of the both compounds diminished from 20 min to 90 min after injection, and that of [11C]UQL was higher (P < 0.05 at 20 min; not significant at 90 min) than that of [11C]UQN. 3.3. Plasma metabolite analysis In the case of UQL administration, plasma concentration of UQL and UQN did not drastically change, and the reduced CoQ10 rate was maintained over 95%, that is, almost all of administered UQL was not oxidized in the body. On the other hand, in the case of UQN administration, plasma concentration of UQN slightly decreased, and that of UQL increased as time passed. In other words, injected UQN was reduced in vivo, and the less injected dose, the higher reduced CoQ10 rate was observed (Fig. 3). 4. Discussion In the present study, we demonstrated the difference of tissue or organ uptake of the reduced and oxidized form of coenzyme Q10 (ubiquinol-10 and ubiquinone-10, respectively) by PET and
Please cite this article as: K. Watanabe et al., PET imaging of 11C-labeled coenzyme Q10: Comparison of biodistribution between [11C]ubiquinol10 and [11C]ubiquinone-10, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.03.073
K. Watanabe et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Fig. 1. Integrated whole-body maximum intensity projection images of PET at the corresponding time periods after injection of [11C]ubiquinol-10 ([11C]UQL, upper figures) or [11C] ubiquinone-10 ([11C]UQN, lower figures). PET studies were performed as described in “Materials and Methods.” By pseudo-color coding, red color shows higher uptake and blue color shows lower uptake of radioactivity. Higher accumulation of [11C]UQL and [11C]UQN were mainly observed in the liver, lung, and spleen. We could also see the difference in the heart and aortas (especially hot-spot in the vicinity of abdominal aortic bifurcation) and head regions including the brain, somewhat higher uptake was seen by [11C]UQL. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2. Biodistribution at 20 min and 90 min after the administration of [11C]ubiquinol-10 or [11C]ubiquinone-10 via a tail vein. According to the experimental procedure described under “Materials and Methods,” we calculated %injected dose per gram of tissue (%ID/g). a, cerebrum; b, cerebellum; c, brown adipose tissue; d, white adipose tissue; e, heart; f, lung; g, liver; h, spleen; i, quadriceps femoris muscle; j, kidney; k, testis; and l, blood. Values are shown as the mean ± standard deviation (n ¼ 6 for each condition). Statistically significant by two-way analysis of variance followed by a post-hoc multiple comparisons test (Bonferroni correction): *, P < 0.05; **, P < 0.01.
biodistribution study in rats. The final goal of the study is the pharmacokinetics of both compounds in human, especially related to the health state and pathological insult. Of course, in the neuroscience field, the energy demand by neural activity and glial
activity by various tasks and neuroinflammation might be delineated by uptake of ubiquinol demanded. Here, we could show the difference between the uptake of UQL and UQN into the brain, especially significantly higher uptake of [11C]UQL into the brain
Please cite this article as: K. Watanabe et al., PET imaging of 11C-labeled coenzyme Q10: Comparison of biodistribution between [11C]ubiquinol10 and [11C]ubiquinone-10, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.03.073
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Fig. 3. Analysis of plasma samples at 2, 7, 20, 90 min after intravenous injection of unlabeled ubiquinol-10 (A) or ubiquinone-10 (B) with different doses: △, 1 mg/kg; ,, 2 mg/kg; B, 4 mg/kg. The amounts of ubiquinol, ubiquinone, total coenzyme Q10, and the ratio of ubiquinol/total coenzyme Q10 (Reduced CoQ10 ratio) were calculated and shown with time course. Values are shown as the mean ± standard error of the mean (n ¼ 3 or 4 for each condition).
(cerebrum and cerebellum), white adipose tissue, muscle, kidney, and testis at 20 and 90 min after injection and into the brown adipose tissue at 90-min time point was observed as compared with those of [11C]UQN. On the contrary, the uptake of the oxidized form [11C]UQN was higher in the spleen. The transporter (kinds and/or kinetics) for uptake and excretion of UQL and UQN might be different, but as far as we know, such transporters have not yet identified in the case of UQL and UQN. Further studies on the transporters for uptake and excretion are needed. We have tried to analyze the metabolites from UQL and UQN injected into rats by thin-layer chromatography, however UQL easily oxidized into UQN on the silica gel, even when we added various reducing agents to the plasma samples. Therefore, in a separate study, we injected unlabeled UQL or UQN into the tail vein of rats, and analyzed by LC-MS/MS. This LC-MS/MS method is not applicable for a number of plasma samples, since the physical halflife of 11C is only 20.4 min, very short. And the injected dose in the metabolite analysis of unlabeled UQL or UQN was ca. 200-fold, 400fold, and 800-fold higher than that of the PET and biodistribution studies using [11C]UQL or [11C]UQN, because of the detection limit of the small amount of metabolites by LC-MS/MS analysis. However, we detected most of main peaks of UQL and UQN. In vivo reduction of UQN occurs according to some limited speed, so that the reduced CoQ10 rate was increased with the smaller dose injection (Fig. 3B). Judged from the study, we could think about almost full conversion of [11C]UQN in the time frame of PET and biodistribution studies. This means that the uptake of [11C]UQL seems to show the true distribution of UQL in the body, but the uptake of the radioactivity originated from [11C]UQN injected contained the proportion of [11C]UQL uptake in the later phase after injection. Therefore, true [11C]UQN uptake into the brain (cerebrum and cerebellum), kidney, testis, muscle and adipose tissues might be lower than the present data (Fig. 2). The difference between uptake of [11C]UQL and [11C]UQN could be much larger. Concerning the metabolism and kinetics of CoQ10, Bentinger et al. [13] reported the fate of 3H-labeled UQN in days order after intraperitoneal administration to rats. They showed higher uptake
into the spleen, liver, and white blood cells, moderate uptake into the adrenals, ovaries, and kidney, lower uptake into the thymus and heart, then into the muscle and brain at 1, 2, 6, 10, 13 days after administration. The distribution data are somehow similar to our present data, but considering from our data, further than one day after the injection, most of [3H]UQN could be converted to [3H]UQL then further metabolized, so that they got the mixed distribution data with UQN injected but UQL converted then metabolized in vivo. They also identified the further metabolites than reduced form of UQN, but at least one day after the injection. The time window is so much different from our experiments. Intrinsic CoQ in the rats is CoQ9 instead of CoQ10 which is in humans and other mammalian species. However, the incorporation or transport between CoQ9 and CoQ10 was thought to be not so different each other, even for NADH: ubiquinone oxidoreductase (Respiratory complex I [14]). In our experiments with unlabeled UQL or UQN load, CoQ9 content in the rat plasma was not so much influenced by CoQ10 load. Therefore, we are thinking that our case with [11C]UQL and [11C]UQN injected as a tracer dose could follow the dynamics of CoQ9 dynamics in the whole body in rats. This is the same as CoQ10 dynamics in humans. Soon after the approval from the Ethics Committee, we would like to start the clinical PET study with [11C]UQL and [11C]UQN, and then step up to human PET study with brain tasks and diseases including neuropsychiatric disorders and developmental disorders. Acknowledgments The authors thank Mr. M. Kurahashi of Sumitomo Heavy Industry Accelerator Service Ltd. for operating the cyclotron. This work was supported in part by an extramural research grant from KANEKA Corporation (Osaka, Japan) to Yasuyoshi Watanabe. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.03.073.
Please cite this article as: K. Watanabe et al., PET imaging of 11C-labeled coenzyme Q10: Comparison of biodistribution between [11C]ubiquinol10 and [11C]ubiquinone-10, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.03.073
K. Watanabe et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
References [1] M. Turunen, J. Olsson, G. Dallner, Metabolism and function of coenzyme Q, Biochim. Biophys. Acta Biomembr. 1660 (2004) 171e199, https://doi.org/ 10.1016/j.bbamem.2003.11.012. [2] G. Lenaz, R. Fato, G. Formiggini, M.L. Genova, The role of Coenzyme Q in mitochondrial electron transport, Mitochondrion 7 (2007) S8eS33, https:// doi.org/10.1016/j.mito.2007.03.009. [3] R.T. Matthews, L. Yang, S. Browne, M. Baik, M.F. Beal, Coenzyme Q10 administration increases brain mitochondrial concentrations and exerts neuroprotective effects, Proc. Natl. Acad. Sci. U. S. A 95 (1998) 8892e8897, https:// doi.org/10.1073/pnas.95.15.8892. [4] M. Beal, Mitochondrial dysfunction and oxidative damage in Alzheimer's and Parkinson's diseases and coenzyme Q10 as a potential treatment, J. Bioenerg. Biomembr. 36 (2004). http://link.springer.com/article/10.1023/B:JOBB. 0000041772.74810.92. [5] M. Spindler, M. Flint Beal, C. Henchcliffe, Coenzyme Q10 effects in neurodegenerative disease, Neuropsychiatric Dis. Treat. 5 (2009) 597e610, https:// doi.org/10.2147/NDT.S5212. [6] M.T. Lin, M.F. Beal, Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases, Nature 443 (2006) 787e795, https://doi.org/10.1038/ nature05292. [7] S. Fukuda, J. Nojima, O. Kajimoto, K. Yamaguti, Y. Nakatomi, H. Kuratsune, Y. Watanabe, Ubiquinol-10 supplementation improves autonomic nervous function and cognitive function in chronic fatigue syndrome, Biofactors 42 (2016) 431e440, https://doi.org/10.1002/biof.1293.
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[8] G. Morris, G. Anderson, M. Berk, M. Maes, Coenzyme Q10 depletion in medical and neuropsychiatric disorders: potential repercussions and therapeutic implications, Mol. Neurobiol. 48 (2013) 883e903, https://doi.org/10.1007/ s12035-013-8477-8. [9] L. Lei, Y. Liu, Efficacy of coenzyme Q10 in patients with cardiac failure: a metaanalysis of clinical trials, BMC Cardiovasc. Disord. 17 (2017) 1e7, https:// doi.org/10.1186/s12872-017-0628-9. [10] G. Balercia, A. Mancini, F. Paggi, L. Tiano, A. Pontecorvi, M. Boscaro, A. Lenzi, G.P. Littarru, Coenzyme Q10 and male infertility, J. Endocrinol. Investig. 32 (2009) 626e632. http://www.ncbi.nlm.nih.gov/pubmed/19509475. [11] M. Goto, A. Nishiyama, T. Yamaguchi, K. Watanabe, K. Fujii, Y. Watanabe, H. Doi, Synthesis of 11C-labeled ubiquinone and ubiquinol via Pd0 -mediated rapid C-[11C]methylation using [11C]methyl iodide and 39-demethyl-39(pinacolboryl)ubiquinone, J. Label. Comp. Radiopharm. 62 (2019) 86e94, https://doi.org/10.1002/jlcr.3700. [12] Y. Tatsuta, K. Kasai, C. Maruyama, Y. Hamano, K. Matsuo, H. Katano, S. Taira, Imaging mass spectrometry analysis of ubiquinol localization in the mouse brain following short-term administration, Sci. Rep. 7 (2017) 1e7, https:// doi.org/10.1038/s41598-017-13257-8. [13] M. Bentinger, G. Dallner, T. Chojnacki, E. Swiezewska, Distribution and breakdown of labeled coenzyme Q10 in rat, Free Radic. Biol. Med. 34 (2003) 563e575, https://doi.org/10.1016/S0891-5849(02)01357-6. [14] J.G. Fedor, A.J.Y. Jones, A. Di Luca, V.R.I. Kaila, J. Hirst, Correlating kinetic and structural data on ubiquinone binding and reduction by respiratory complex I, Proc. Natl. Acad. Sci. Unit. States Am. 114 (2017) 12737e12742, https:// doi.org/10.1073/pnas.1714074114.
Please cite this article as: K. Watanabe et al., PET imaging of 11C-labeled coenzyme Q10: Comparison of biodistribution between [11C]ubiquinol10 and [11C]ubiquinone-10, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.03.073
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
PET imaging of 11C-labeled coenzyme Q10: Comparison of biodistribution between [11C]ubiquinol-10 and [11C]ubiquinone-10 Kyosuke Watanabe a, b, c, d, Satoshi Nozaki a, b, Miki Goto a, b, Ken-ichi Kaneko a, b, Emi Hayashinaka a, b, Satsuki Irie a, b, Akira Nishiyama e, Kazuaki Kasai f, Kenji Fujii e, Yasuhiro Wada a, b, Kei Mizuno a, b, c, d, Kenji Mizuseki d, Hisashi Doi a, b, Yasuyoshi Watanabe a, b, c, * a
RIKEN Center for Life Science Technologies, Japan RIKEN Center for Biosystems Dynamics Research, Japan c RIKEN Compass to Healthy Life Research Complex Program, Kobe, Hyogo, Japan d Osaka City University Graduate School of Medicine, Osaka, Japan e KANEKA Corporation, Osaka, Japan f Takasago Analysis Center, Kaneka Techno Research Corporation, Takasago, Hyogo, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 March 2019 Accepted 13 March 2019 Available online xxx
Coenzyme Q10 (CoQ10) plays a key role not only as an essential electron carrier in the mitochondrial electron transport chain, but also as an antioxidant to protect cells from oxidative stress. CoQ10 supplementation is expected to be effective for a variety of diseases. The predominant forms of CoQ10 are the ubiquinol-10 (reduced form) and ubiquinone-10 (oxidized form). Both forms of CoQ10 supplements are commercially available, however, their kinetic difference is still unclear. In order to conduct in vivo analysis of the kinetics of ubiquinol-10 and ubiquinone-10, we succeeded in synthesizing 11C-labeled ubiquinol-10 ([11C]UQL) and ubiquinone-10 ([11C]UQN), respectively. In the present study, we aimed to investigate the kinetics of [11C]UQL and [11C]UQN, both of which were administered via the tail vein of 8week-old male Sprague-Dawley rats. Whole-body positron emission tomography (PET) imaging was performed to follow the time course of accumulation in the liver, spleen, brain, and other organs. Then, at the two typical time points at 20 or 90 min after injection, we conducted the biodistribution study. Various organs/tissues and blood were collected, weighed and counted with a gamma counter. Percent injected dose per gram of tissue (%ID/g) was calculated as the indicator of the accumulation of each compound. As the results, at both time points, %ID/g of [11C]UQL in the cerebrum, cerebellum, white adipose tissue, muscle, kidney, and testis were higher (P < 0.05) than that of [11C]UQN: at 90-min time point, %ID/g of [11C]UQL in the brown adipose tissue was higher (P < 0.05) than that of [11C]UQN: on the contrary, %ID/g of [11C]UQL in the spleen was lower (P < 0.05) than that of [11C]UQN at 90 min. In a separate study of the metabolite analysis in the plasma, UQL injected into the tail vein of rats was almost unchanged during the PET scanning time, but UQN was gradually converted to the reduced form UQL. Therefore, the uptake values of UQL into the tissues and organs were rather accurate but those of UQN might be the sum of UQN uptake and partly converted UQL uptake. These studies suggested that the accumulation level of administered CoQ10 differs depending on its redox state, and that CoQ10 redox state could be crucial for optimization of the effective supplementation. © 2019 Elsevier Inc. All rights reserved.
Keywords: Coenzyme Q10 Ubiquinol Ubiquinone PET Biodistribution Metabolism
1. Introduction
* Corresponding author. RIKEN Compass to Healthy Life Research Complex Program, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan. E-mail addresses: [email protected] (K. Watanabe), [email protected] (Y. Watanabe).
Coenzyme Q10 (CoQ10) plays a key role not only as an essential electron carrier in the mitochondrial electron transport chain, but also as an anti-oxidant to protect cells from oxidative stress [1,2]. CoQ10 supplementation is expected to be effective for a variety of
https://doi.org/10.1016/j.bbrc.2019.03.073 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: K. Watanabe et al., PET imaging of 11C-labeled coenzyme Q10: Comparison of biodistribution between [11C]ubiquinol10 and [11C]ubiquinone-10, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.03.073
2
K. Watanabe et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
diseases such as neurodegenerative disease [3e6], neuropsychiatric disorders [7], myalgic encephalomyelitis/chronic fatigue syndrome [8], cardiovascular insults [9], and male infertility [10]. The predominant forms of CoQ10 are the ubiquinol-10 (reduced form) and ubiquinone-10 (oxidized form). Both forms of CoQ10 supplements are commercially available, however, their kinetic difference is still unclear. In order to conduct in vivo analysis of the kinetics of ubiquinol-10 and ubiquinone-10 not only in animals but also in human, we succeeded in synthesizing 11C-labeled ubiquinol-10 ([11C]UQL) and ubiquinone-10 ([11C]UQN), respectively [11]. In the present study, we aimed to investigate the difference of kinetics of [11C]UQL and [11C]UQN injected intravenously.
2. Materials and methods 2.1. Experimental animals Male Sprague-Dawley rats (8e9 weeks old) were sourced from CLEA Japan, Inc. (Tokyo, Japan). They were housed in a light- and temperature-controlled environment with unlimited access to food and water available. All experimental protocols were approved by the Ethics Committee on Animal Care and Use of RIKEN and were performed in accordance with the National Institute of Health guide for the care and use of Laboratory animals.
2.5. Plasma metabolite analysis The reduced form UQL is easily oxidized onto the thin-layer chromatography, possibly by activated silica. Therefore, in this case, we could not conduct the radio-metabolite analysis with rapid analytical methods routinely used in most of PET study. We chose the HPLC methods with longer time consumption, so that in place of labeled compounds we injected the unlabeled UQL or UQN into the rats and took plasma samples (n ¼ 3 or 4 for each condition) with time for LC-MS/MS analyses [12]. UQL and UQN concentration in the plasma was measured using LC-MS/MS equipped with a tandem mass spectrometer at Kaneka Techno Research Corporation. Briefly, 0.7 mL of extraction solvent was added to 0.1 mL of plasma and mixed. After centrifugation, the supernatant was filtered through a membrane filter and used as a sample for LC-MS/ MS. Measurements were made using a QTRAP® 5500 LC-MS/MS system (AB Sciex, Framingham, Mass., USA). 2.6. Statistical analysis Two-way analysis of variance followed by a post-hoc multiple comparisons test (Bonferroni correction) was performed using GraphPad Prism 6 (GraphPad Software, CA, USA). P values of less than 0.05 were considered statistically significant. 3. Results
2.2. Synthesis of
11
C-labeled ubiquinone-10 and ubiquinol-10
Carbon-11-labeled ubiquinonol-10 and ubiquinone-10 were synthesized by the method recently developed and reported by our group [11]. The specification of these labeled compounds provided for the present study were as follows: The molar activity and injected mass of [11C]UQL and [11C]UQN at the time of intravenous administration to rats were 66.7 ± 6.8 GBq/mmol (1.08 ± 0.11 nmol) and 55.2 ± 10.3 GBq/mmol (1.31 ± 0.18 nmol) (mean ± SD), respectively. The radiochemical purity and chemical purity of [11C]UQL were 97.5 ± 1.7% and 77.8 ± 7.8%, and those of [11C]UQN were 96.8 ± 3.2% and 96.7 ± 2.7%, respectively. 2.3. Positron emission tomography (PET) imaging The rats were anesthetized with 1.5% isoflurane and the cannula to each rat was inserted into their tail vein. They were placed on the PET scanner (microPET Focus 220; Siemens, Knoxville, TN, USA) and injected the radiolabeled compound (ca. 75 MBq per animal) via the tail vein. Emission data were acquired for 90 min using the continuous bed motion acquisition method. Following acquisition, the emission data were sorted into a 2D data set using Fourierrebinning algorithm and reconstructed with a maximum likelihood expectation maximization algorithm.
2.4. Biodistribution studies At 20 min and 90 min after the administration of [11C]UQL or [11C]UQN via a tail vein, the rats were sacrificed, and tissues of interest, blood and urine were collected and weighed (n ¼ 6 for each condition). Their radioactivity was measured using the scintillation counter (Wallac Wizard 1480/2480; PerkinElmer, Waltham, MA, USA). Tissues collected were cerebrum, cerebellum, heart, lung, liver, spleen, kidney, pancreas, quadriceps femoris muscle, brown adipose tissue, white adipose tissue, and testis. Percent injected dose per gram of tissue (%ID/g) was calculated as the indicator of the accumulation of the injected compound.
3.1. PET imaging Integrated whole-body maximum intensity projection images at the corresponding time periods after injection are successfully visualized (Fig. 1). Accumulation of [11C]UQL and [11C]UQN were mainly observed in the liver, lung, and spleen. We could also see the difference in the heart and aortas and head regions including the brain, somewhat higher uptake was seen by [11C]UQL. 3.2. Biodistribution studies As shown in Fig. 2, at both time points (20 and 90 min after injection), %ID/g of [11C]UQL in the cerebrum, cerebellum, white adipose tissue, muscle, kidney, and testis were higher (P < 0.05) than that of [11C]UQN, whereas that of [11C]UQL in the spleen at 90 min was lower (P < 0.05) than that of [11C]UQN. %ID/g of [11C] UQL in the brown adipose tissue was higher (P < 0.05) than that of [11C]UQN at 90 min after injection. Blood concentration of the both compounds diminished from 20 min to 90 min after injection, and that of [11C]UQL was higher (P < 0.05 at 20 min; not significant at 90 min) than that of [11C]UQN. 3.3. Plasma metabolite analysis In the case of UQL administration, plasma concentration of UQL and UQN did not drastically change, and the reduced CoQ10 rate was maintained over 95%, that is, almost all of administered UQL was not oxidized in the body. On the other hand, in the case of UQN administration, plasma concentration of UQN slightly decreased, and that of UQL increased as time passed. In other words, injected UQN was reduced in vivo, and the less injected dose, the higher reduced CoQ10 rate was observed (Fig. 3). 4. Discussion In the present study, we demonstrated the difference of tissue or organ uptake of the reduced and oxidized form of coenzyme Q10 (ubiquinol-10 and ubiquinone-10, respectively) by PET and
Please cite this article as: K. Watanabe et al., PET imaging of 11C-labeled coenzyme Q10: Comparison of biodistribution between [11C]ubiquinol10 and [11C]ubiquinone-10, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.03.073
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Fig. 1. Integrated whole-body maximum intensity projection images of PET at the corresponding time periods after injection of [11C]ubiquinol-10 ([11C]UQL, upper figures) or [11C] ubiquinone-10 ([11C]UQN, lower figures). PET studies were performed as described in “Materials and Methods.” By pseudo-color coding, red color shows higher uptake and blue color shows lower uptake of radioactivity. Higher accumulation of [11C]UQL and [11C]UQN were mainly observed in the liver, lung, and spleen. We could also see the difference in the heart and aortas (especially hot-spot in the vicinity of abdominal aortic bifurcation) and head regions including the brain, somewhat higher uptake was seen by [11C]UQL. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2. Biodistribution at 20 min and 90 min after the administration of [11C]ubiquinol-10 or [11C]ubiquinone-10 via a tail vein. According to the experimental procedure described under “Materials and Methods,” we calculated %injected dose per gram of tissue (%ID/g). a, cerebrum; b, cerebellum; c, brown adipose tissue; d, white adipose tissue; e, heart; f, lung; g, liver; h, spleen; i, quadriceps femoris muscle; j, kidney; k, testis; and l, blood. Values are shown as the mean ± standard deviation (n ¼ 6 for each condition). Statistically significant by two-way analysis of variance followed by a post-hoc multiple comparisons test (Bonferroni correction): *, P < 0.05; **, P < 0.01.
biodistribution study in rats. The final goal of the study is the pharmacokinetics of both compounds in human, especially related to the health state and pathological insult. Of course, in the neuroscience field, the energy demand by neural activity and glial
activity by various tasks and neuroinflammation might be delineated by uptake of ubiquinol demanded. Here, we could show the difference between the uptake of UQL and UQN into the brain, especially significantly higher uptake of [11C]UQL into the brain
Please cite this article as: K. Watanabe et al., PET imaging of 11C-labeled coenzyme Q10: Comparison of biodistribution between [11C]ubiquinol10 and [11C]ubiquinone-10, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.03.073
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Fig. 3. Analysis of plasma samples at 2, 7, 20, 90 min after intravenous injection of unlabeled ubiquinol-10 (A) or ubiquinone-10 (B) with different doses: △, 1 mg/kg; ,, 2 mg/kg; B, 4 mg/kg. The amounts of ubiquinol, ubiquinone, total coenzyme Q10, and the ratio of ubiquinol/total coenzyme Q10 (Reduced CoQ10 ratio) were calculated and shown with time course. Values are shown as the mean ± standard error of the mean (n ¼ 3 or 4 for each condition).
(cerebrum and cerebellum), white adipose tissue, muscle, kidney, and testis at 20 and 90 min after injection and into the brown adipose tissue at 90-min time point was observed as compared with those of [11C]UQN. On the contrary, the uptake of the oxidized form [11C]UQN was higher in the spleen. The transporter (kinds and/or kinetics) for uptake and excretion of UQL and UQN might be different, but as far as we know, such transporters have not yet identified in the case of UQL and UQN. Further studies on the transporters for uptake and excretion are needed. We have tried to analyze the metabolites from UQL and UQN injected into rats by thin-layer chromatography, however UQL easily oxidized into UQN on the silica gel, even when we added various reducing agents to the plasma samples. Therefore, in a separate study, we injected unlabeled UQL or UQN into the tail vein of rats, and analyzed by LC-MS/MS. This LC-MS/MS method is not applicable for a number of plasma samples, since the physical halflife of 11C is only 20.4 min, very short. And the injected dose in the metabolite analysis of unlabeled UQL or UQN was ca. 200-fold, 400fold, and 800-fold higher than that of the PET and biodistribution studies using [11C]UQL or [11C]UQN, because of the detection limit of the small amount of metabolites by LC-MS/MS analysis. However, we detected most of main peaks of UQL and UQN. In vivo reduction of UQN occurs according to some limited speed, so that the reduced CoQ10 rate was increased with the smaller dose injection (Fig. 3B). Judged from the study, we could think about almost full conversion of [11C]UQN in the time frame of PET and biodistribution studies. This means that the uptake of [11C]UQL seems to show the true distribution of UQL in the body, but the uptake of the radioactivity originated from [11C]UQN injected contained the proportion of [11C]UQL uptake in the later phase after injection. Therefore, true [11C]UQN uptake into the brain (cerebrum and cerebellum), kidney, testis, muscle and adipose tissues might be lower than the present data (Fig. 2). The difference between uptake of [11C]UQL and [11C]UQN could be much larger. Concerning the metabolism and kinetics of CoQ10, Bentinger et al. [13] reported the fate of 3H-labeled UQN in days order after intraperitoneal administration to rats. They showed higher uptake
into the spleen, liver, and white blood cells, moderate uptake into the adrenals, ovaries, and kidney, lower uptake into the thymus and heart, then into the muscle and brain at 1, 2, 6, 10, 13 days after administration. The distribution data are somehow similar to our present data, but considering from our data, further than one day after the injection, most of [3H]UQN could be converted to [3H]UQL then further metabolized, so that they got the mixed distribution data with UQN injected but UQL converted then metabolized in vivo. They also identified the further metabolites than reduced form of UQN, but at least one day after the injection. The time window is so much different from our experiments. Intrinsic CoQ in the rats is CoQ9 instead of CoQ10 which is in humans and other mammalian species. However, the incorporation or transport between CoQ9 and CoQ10 was thought to be not so different each other, even for NADH: ubiquinone oxidoreductase (Respiratory complex I [14]). In our experiments with unlabeled UQL or UQN load, CoQ9 content in the rat plasma was not so much influenced by CoQ10 load. Therefore, we are thinking that our case with [11C]UQL and [11C]UQN injected as a tracer dose could follow the dynamics of CoQ9 dynamics in the whole body in rats. This is the same as CoQ10 dynamics in humans. Soon after the approval from the Ethics Committee, we would like to start the clinical PET study with [11C]UQL and [11C]UQN, and then step up to human PET study with brain tasks and diseases including neuropsychiatric disorders and developmental disorders. Acknowledgments The authors thank Mr. M. Kurahashi of Sumitomo Heavy Industry Accelerator Service Ltd. for operating the cyclotron. This work was supported in part by an extramural research grant from KANEKA Corporation (Osaka, Japan) to Yasuyoshi Watanabe. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.03.073.
Please cite this article as: K. Watanabe et al., PET imaging of 11C-labeled coenzyme Q10: Comparison of biodistribution between [11C]ubiquinol10 and [11C]ubiquinone-10, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.03.073
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Please cite this article as: K. Watanabe et al., PET imaging of 11C-labeled coenzyme Q10: Comparison of biodistribution between [11C]ubiquinol10 and [11C]ubiquinone-10, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.03.073