{"paper_id":"1163bd4d-55d0-4d06-b88a-4f2a05a1d56b","body_text":"Hyperpolarized Multi-organ Spectroscopy of Liver and Brain using 1- 13C-Pyruvate Enhanced via Para-Hydrogen | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hyperpolarized Multi-organ Spectroscopy of Liver and Brain using 1- 13C-Pyruvate Enhanced via Para-Hydrogen Theresa L. K. Hune, Salvatore Mamone, Andreas B. Schmidt, Inês Mahú, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2919623/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Aug, 2023 Read the published version in Applied Magnetic Resonance → Version 1 posted 7 You are reading this latest preprint version Abstract Hyperpolarization in nuclear magnetic resonance boosts the signals by several orders of magnitude. Using the singlet spin order of para-hydrogen to create large non-equilibrium spin polarization is a fast approach to obtain hyperpolarized metabolites in seconds. In recent years, it has attracted particular interest in the field of biomedicine because signal-enhanced and 13 C-enriched metabolites allow for real-time metabolic investigations in combination with imaging in vivo. With this, metabolism can be traced and characterized with spatial selectivity in the body. Here, we introduce a method to use signal-enhanced metabolites to study multiple organs in separate injections to obtain real-time kinetics in vivo of these organs. Using hyperpolarized 1- 13 C-pyruvate, we measured the kinetics of the conversion from pyruvate to lactate in the brain and the liver of mice. This we did by injecting the hyperpolarized pyruvate two times within half an hour and using each injection to measure the spectra of one region of interest. Organ cross-talk and especially how different organs affect each other in diseases is of major interest and rarely understood, because of the high complexity of biological systems. With the proof-of-principle study provided here, we are introducing a new tool to study organ-related interaction in vivo. It allows the characterization of different organs of the same animal at the same time point, which is enabled by the fast signal enhancement achieved with para-hydrogen. Figures Figure 1 Figure 2 Figure 3 1 Introduction Nuclear Magnetic Resonance (NMR) Spectroscopy has a high diagnostic value and allows for the detection of metabolites and characterization of metabolism in vivo [ 1 – 4 ]. However, it also suffers from low sensitivity, and the tracking of metabolic kinetics in vivo is not possible with standard approaches that require a large number of averages to obtain signals above the noise level. To overcome this challenge, hyperpolarization techniques were introduced enhancing NMR signals by over four orders of magnitude [ 5 ]. Such large signal enhancements enable the study of metabolic real-time kinetic events in vivo. In order to do this, heteronuclei, like 13 C are typically used, since they have the advantage of a longer relaxation time T 1 than protons, which allows for longer traceability of the metabolite in vivo – for 13 C typically up to several minutes [ 6 ]. There are different approaches to achieve hyperpolarization. Of those, dissolution Dynamic Nuclear Polarization (DNP) is the most prominent hyperpolarization technique for studying metabolism in vivo. DNP uses electron polarization to enhance the signals of close nuclear spins [ 7 – 9 ]. It has been used in clinical trials and produces highly polarized metabolites in high quantities [ 10 – 15 ]. However, it is a slow technique, taking tens of minutes to hours to produce hyperpolarized molecules, which appears to be a significant barrier to translation into clinical practice which was tried to circumvent by polarizing up to four samples at the same time [ 16 , 17 ]. Para-hydrogen (pH 2 ) induced polarization (PHIP) is a comparably new technique in preclinical applications and offers the possibility to prepare hyperpolarized metabolites in a much shorter amount of time. In pH 2 nearly all hydrogen molecules occupy the same spin state, offering a high degree of spin order [ 18 – 21 ]. This spin order is transferred to the molecule of interest by a method, which is called Para-Hydrogen Induced Polarization by means of Side-Arm Hydrogenation (PHIP-SAH) [ 22 – 32 ]. The general workflow is depicted in Fig. 1 . A precursor, in which the molecule of interest is linked to an unsaturated side-arm via a labile bond, is hydrogenated using pH 2 and a catalyst in acetone. Afterwards, the spin order from the hydrogen nuclei is transferred to the 13 C of interest using a series of carefully timed radiofrequency pulses called the MINERVA (Maximizing Insensitive Nuclei Enhancement Reached Via para-hydrogen Amplification) sequence [ 33 ]. After the transfer, the side-arm is cleaved by addition of a basic water solution. After a rapid evaporation of the acetone, the remaining aqueous solution is worked-up by addition of a buffer to adjust the pH to physiological conditions and filtration of the hydrogenation catalyst – leading to a clean solution of the hyperpolarized metabolite in water, which can then be injected into an organism. The whole procedure from the start of hydrogenation to the injection requires typically about one minute. Once injected, the hyperpolarized spin label is visible in the NMR spectra and can be used for the localization and characterization of disease or the investigation of metabolism in vivo. [ 34 – 39 ] The spectra of the metabolites can either be measured over the whole body [ 40 , 41 ] or selectively over a region of interest, for example a single organ [ 42 , 43 ]. However, usually a choice has to be made as to which region should be observed, because the allowed injection volume and therefore the sensitivity of the experiment is limited especially in preclinical studies performed on rodents. Multiple injections would provide a way to work around this limitation. In vivo studies with DNP in rodents (mostly rats) demonstrated that two successive injections can be used to study the same region of interest, as to get two data points from one animal [ 44 – 46 ]. Studying large cohorts however appears to be out of reach even with multiple samples being enhanced at the same time. This work demonstrates the possibility to study two different organs of the same animal with PHIP, using two injections of hyperpolarized 1- 13 C-pyruvate. We recorded spectra of the region of the brain and the liver of mice within less than half an hour and analyzed them with regard to the conversion of pyruvate to lactate. This study demonstrates that the high throughput of hyperpolarized metabolites achieved with para-hydrogen enables extensive studies of complex biological systems. With this, metabolism in different organs of the same animal can be studied simultaneously, opening possibilities to study organ crosstalk and reduce the necessary number of animals by accessing more information from each one. The method is minimally invasive, the animals can be woken up again and used for further studies, enabling also longitudinal characterization of metabolism and disease progression. 2 Methods 2.1 In situ production of para-hydrogen Molecular hydrogen was produced by an electrolysis unit (H2PEM-510, Parker Hannifin, Cleveland, Ohio) and fed directly into a para-hydrogen generator (BPHG 90, Bruker Corporation, Ettlingen, Germany) at 36 K. The para-hydrogen was compressed to a pressure of 10 bar in a 1L buffer cylinder and led to the sample through a home-built tubing system. 2.2 Hyperpolarization All chemicals except the precursor were purchased from commercial suppliers and used as received. For the samples, a solution of 13 mM Rhodium catalyst, [1,4- Bis(diphenylphosphino)butane](1,5-cyclooctadiene)rhodium(I) tetrafluoroborate, (Sigma Aldrich 79255-71-3, M = 724.4 g/mol) and 95 mM of the precursor 3-(phenyl-d5)prop-2-yn-1-yl-2-13C-1,1-d2 2-oxopropanoate-1-13C in acetone-d6 was prepared. For each sample, 200 µL of this solution were filled into an 8-inch 5 mm NMR tube. The hyperpolarization procedure was performed using a home-built electromagnet operating at 21.5 mT. The samples were degassed by bubbling nitrogen gas through the solution prewarmed in a water bath at 80°C for about two minutes. Afterwards, the NMR tubes were placed inside a custom-built dual channel probe in the bore of the electromagnet for the PHIP experiments. A more detailed description of the system is given in [ 27 , 31 ]. Para-hydrogen was bubbled through the sample at a pressure of 7 bar for 15 seconds. Following the hydrogenation, the MINERVA pulse sequence was used to transfer the polarization from the protons to the 13 C of interest, in this case the 1- 13 C of the pyruvate moiety [ 33 ]. After pressure release, a solution of 100 mM Na 2 CO 3 in water was added to cleave the ester bond. The acetone was removed by evaporation through flushing with nitrogen gas at a pressure of 8 bar and the pH was adjusted to physiological conditions using 1X PBS-buffered saline at pH 1.1. After passing the sample through a membrane filter with a 1.0 µm pore size to remove the catalyst, the aqueous solution containing clean hyperpolarized pyruvate was ready to be injected. 2.3 Animal Housing and Mouse Handling Experiments were carried out in accordance with the EU directive 2010/63/EU for animal experiments and the German Animal Welfare Act (TierSchG, 2006) and were approved by the regional authorities (LANUV NRW; application number 81-02.04.2020.A157). Animals used in this study were housed under standard laboratory conditions in a 12 h light cycle with food and water available ad libitum and kept in groups of up to five. The mice were between three and four months old and fed a high-fat diet. Male mice (n = 3) with a C57BL/6 N genetic background were used for the measurements. The mice were anesthetized in a dedicated container using 4% isoflurane in O 2 and N 2 O (1:3). Afterwards, the isoflurane was decreased and kept between 2% and 3% for the whole measurement. A tail vein catheter was placed and the mice were positioned head-prone on a custom-built warming water mat to ensure constant body temperature. Body temperatures as well as breathing rates were monitored during the anesthesia. The mice were positioned inside the coil alongside a urea phantom, used as a reference for the 13 C chemical shift scale, so that both the brain and the liver were inside the sensitive volume. 2.4 MR Measurements All measurements were performed in a 9.4 T preclinical Bruker MR scanner (Bruker Corporation, Ettlingen, Germany) equipped with a 13C/1H Volume Coil by RAPID Biomedical (RAPID Biomedical GmbH, Rimpar, Germany). Localized spectroscopy of the brain region and the liver region were measured with a standard spectroscopy sequence from Bruker (NSPECT) with axial slice selection. The slices were positioned using anatomical proton images for reference, with a slice thickness for the brain of 18 mm and for the liver of 21 mm. For each measurement, a series of 128 spectra were recorded with a flip angle of 18 ° and TR = 2 s, resulting in a measurement time of about four minutes. The acquisition was started prior to injection to record the increase of the NMR signal following pyruvate uptake in the organ of interest. The reference power for protons was adjusted automatically. The reference power for carbon was determined to be 0.19 W. The pulse duration for the spectroscopy on carbon was set to 0.2 ms. The anatomical proton images were recorded using a Bruker standard FLASH sequence with coronal and sagittal slice orientation. The number of slices was 70 with a slice thickness of 1 mm and the matrix size was 256 x 256 in a 80 mm x 80 mm FOV. Each mouse was injected twice with independently generated hyperpolarized substrate. For each measurement, 2.5 µL/g of body weight of pyruvate solution (40 mM) was injected through a catheter into the lateral tail vein of the mouse. The first measurement was performed on the brain, and the second on the liver. The time between the two injections was kept below half an hour. Each mouse was kept no longer than 2 hours under anesthesia. After the experiment, the mice were woken up. 2.5 Data Analysis The spectra were processed using TopSpin 4.0. Phase-correction as well as line-broadening and baseline-correction were applied. All further analysis was performed using Python 3.0 in Jupyter Notebooks. In each spectrum, the peaks of pyruvate and lactate were integrated and summed up to determine the area under the curves (AUCs). From the ratio of the AUCs, the conversion coefficient k PL was calculated according to [ 47 ], under the assumption that the back-conversion is negligible ( k LP =0). In order to do this, the T 1 of pyruvate and lactate were estimated to be T 1 (Pyr) = 25 s, T 1 (Lac) = 20 s. 3 Results & Discussion Figure 2 shows the spectrum of the 13 C-NMR spectrum of the hyperpolarized pyruvate, measured in the low-field spectrometer directly after para-hydrogen bubbling and application of the polarization transfer sequence. Below the pyruvate spectrum, the 1 H-spectrum of water, acquired at the same Larmor Frequency (~ 230 kHz) using 2000 scans is shown. Compared to thermal polarization, the signal of the pyruvate is enhanced by a factor of 7.7 million, which corresponds to a 13 C polarization of 14.6% at 21.5 mT. After the full workup procedure, the concentration of the pyruvate in physiologically adjusted neat water solutions was found to be ~ 40 mM with polarization levels ~ 6.5%. This biocompatible solution was then rapidly transferred to the MRI scanner and administered into the waiting mouse. Each mouse was injected twice with freshly hyperpolarized pyruvate solutions produced on time. The first injection was used to record a series of spectra of the brain, while the second one was used to record a series of spectra over the liver region. Figure 3 A shows the positioning of the slices for one representative mouse. After injection, the influx of pyruvate became quickly visible in the spectra. Shortly afterward, lactate production was observed by the appearance of the corresponding signal. From the recorded spectra, the pyruvate as well as the lactate peaks were integrated and the curves plotted in Fig. 3 B and 3 C. In metabolic MR studies, the ratio of the area under the curves (AUC) for the pyruvate and the lactate peak is an important parameter frequently used to characterize metabolism. This ratio correlates with the conversion rate k PL of pyruvate to lactate in the observed slice [ 47 ]. The AUCs were determined from the curves and the conversion rates calculated. The calculated conversion rates were then used to fit the lactate curve. The ratio of the AUCs as well as the k PL in both organs including their standard deviations, were determined from the recorded spectra as summarized in Table 1 . Table 1 Mean and standard deviations for the ratio of the AUCs from lactate and pyruvate as well as the conversion coefficient k PL determined from the spectra. AUC(Lac)/AUC(Pyr) k PL [s − 1 ] Brain 0,58 ± 0,03 0,044 ± 0,003 Liver 0,38 ± 0,22 0,028 ± 0,017 The values suggest the tendency of a lower lactate production in the liver than in the brain. As the mice were young (3–4 months), the low lactate levels are not surprising. However, the pyruvate to lactate conversion in the brain is strongly influenced by the anesthetics, i.e. isoflurane in our case. Shortly after the initiation of the narcosis, the lactate level in the brain has been shown to stabilize, but at a significantly higher level [ 48 , 49 ]. This explains the elevated pyruvate to lactate conversion observed in the brain. Overall, the signal enhancement and the high throughput of the method enable subsequent injections into the same animal in a short amount of time. Sufficient signal intensities are still maintained even though the maximum possible injection volume was distributed between two injections. 4 Conclusions In this work, we used two injections to successfully investigate the metabolism of two different organs in vivo using PHIP-hyperpolarized 1- 13 C-pyruvate. The presented results demonstrate the feasibility of this method for multi-organ studies. In particular, we believe that multiple injections have the advantage over double excitation experiments with one injection that organs can be monitored without accounting for effects due to the saturation of pulses while another organ may be investigated. Additionally, changes in pyruvate-to-lactate conversion rates are a biomarker for diagnosis, therapy and treatment response in several diseases and is hence of high interest. The presented procedure offers the possibility to investigate dependencies and cross-correlations between different regions in the body of the same animal in vivo. It paves the way for the characterization of more complex co-dependencies in disease models, also with regard to longitudinal studies. Additionally, it provides a means to reduce the number of animals used in biological studies, where often large cohorts are necessary to achieve statistical significance and makes them available by increasing the amount of available information from one animal. Additionally, it may prove advantageous to couple the presented approach with a quasi-continuous production of contrast agents [ 50 ]. We would like to emphasize that we see the use of para-hydrogen enhanced metabolites as key to enable biological studies on large cohorts since metabolites can be rapidly generated and multiple investigations on various organs and study subjects can be carried out per day. Declarations Acknowledgements The authors thank Vera Jörke and Dr. Andreas Schmid from WSIC in Tübingen for their help with the warming mat, as well as Ulla Uhlenküken for the support regarding the MR scanner. Furthermore, we thank Dr. Vladimir Belov and Jan Seikowski from the synthesis facility of the MPI-NAT as well as Mario Lengauer and Christian Klaba from the workshop for fine mechanics of the MPI-NAT. S.M. thanks Dr. Lukas Kaltschnee (MPI-NAT) for useful discussions about the para-hydrogen setup. This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant agreement No. 949180). S.G. acknowledges funding by the Max Planck Society and the DFG 495627437. ABS acknowledges funding support by the German Cancer Consortium (DKTK), and the DFG (#SCHM 3694/1-1, #SCHM 3694/2-1, #SFB1479). Material preparation, data collection and analysis were performed by Theresa L. K. Hune, Salvatore Mamone, Inês Mahú and Natascha D’Apolito. S.G. conceived the study. The first draft of the manuscript was written by Theresa L. K. Hune and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Ethical Approval Experiments were carried out in accordance with the EU directive 2010/63/EU for animal experiments and the German Animal Welfare Act (TierSchG, 2006) and were approved by the regional authorities (LANUV NRW; application number 81-02.04.2020.A157). Competing interests The authors declare no competing financial interests Author’s contributions Material preparation, data collection and analysis were performed by Theresa L. K. Hune, Salvatore Mamone, Inês Mahú and Natascha D’Apolito. S.G. conceived the study. The first draft of the manuscript was written by Theresa L. K. Hune and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant agreement No. 949180). S.G. acknowledges funding by the Max Planck Society and the DFG 495627437. ABS acknowledges funding support by the German Cancer Consortium (DKTK), and the DFG (#SCHM 3694/1-1, #SCHM 3694/2-1, #SFB1479). Availability of data and materials All data to support the findings is presented in the manuscript and is made available by the authors upon request. References S. W. Provencher, Estimation of metabolite concentrations from localized in vivo proton NMR spectra. Magn. Reson. Med. 30, 672-679 (1993). M. Hajek, M. 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Cite Share Download PDF Status: Published Journal Publication published 09 Aug, 2023 Read the published version in Applied Magnetic Resonance → Version 1 posted Editorial decision: Major revision 28 May, 2023 Reviews received at journal 26 May, 2023 Reviewers agreed at journal 16 May, 2023 Reviewers invited by journal 15 May, 2023 Editor assigned by journal 13 May, 2023 Submission checks completed at journal 12 May, 2023 First submitted to journal 11 May, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-2919623\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":200031238,\"identity\":\"9f8625a8-8295-4b11-93e4-1d0f057b826e\",\"order_by\":0,\"name\":\"Theresa L. K. Hune\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Max Planck Institute for Multidisciplinary Sciences\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Theresa\",\"middleName\":\"L. K.\",\"lastName\":\"Hune\",\"suffix\":\"\"},{\"id\":200031242,\"identity\":\"bda507f4-ffff-47df-8290-5b20adfeed90\",\"order_by\":1,\"name\":\"Salvatore Mamone\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Max Planck Institute for Multidisciplinary Sciences\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Salvatore\",\"middleName\":\"\",\"lastName\":\"Mamone\",\"suffix\":\"\"},{\"id\":200031244,\"identity\":\"b3cc0c7f-9ec4-49a6-8d9c-4d1fdf56a8d1\",\"order_by\":2,\"name\":\"Andreas B. Schmidt\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ)\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Andreas\",\"middleName\":\"B.\",\"lastName\":\"Schmidt\",\"suffix\":\"\"},{\"id\":200031247,\"identity\":\"b7fa9227-9851-4499-ad92-c37938cbc418\",\"order_by\":3,\"name\":\"Inês Mahú\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Max Planck Institute for Metabolism Research\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Inês\",\"middleName\":\"\",\"lastName\":\"Mahú\",\"suffix\":\"\"},{\"id\":200031248,\"identity\":\"4c2522e5-620d-4b6d-81fd-02ed3d51d7ba\",\"order_by\":4,\"name\":\"Natascha D’Apolito\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Max Planck Institute for Metabolism Research\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Natascha\",\"middleName\":\"\",\"lastName\":\"D’Apolito\",\"suffix\":\"\"},{\"id\":200031249,\"identity\":\"1f6f809c-9245-419b-a008-fff13d568853\",\"order_by\":5,\"name\":\"Dirk Wiedermann\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Max Planck Institute for Metabolism Research\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Dirk\",\"middleName\":\"\",\"lastName\":\"Wiedermann\",\"suffix\":\"\"},{\"id\":200031250,\"identity\":\"a2183396-80cf-4a73-a10e-4429a0d08bf8\",\"order_by\":6,\"name\":\"Jens Brüning\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Max Planck Institute for Metabolism Research\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jens\",\"middleName\":\"\",\"lastName\":\"Brüning\",\"suffix\":\"\"},{\"id\":200031251,\"identity\":\"7c7ffbed-ed95-427d-859c-eb3f40b5d7a5\",\"order_by\":7,\"name\":\"Stefan Glöggler\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABTElEQVRIie2RP0vDQBTAXwlcl4tZI9X6CYQngYhY0q+SEmiXajt26NAQuE46F1LwW4hjwkGmCE4ixKHFSajQopQKWr00VoREwU0wP7g/8O737j0eQE7OH2UabxuraxtAEYf3GaMZ7wkUBskZgwCbvZWCv1DQW9++UXbOHL/3fAEGKzr8/gkrLe360uft7gvsuv0rmHRSCgakZp+EYDEa1A+3sH5wHh2bfBAg6LdhuzAM0wqh+yOZgUXUpq6pyFGPmsgpEcpN05REKFUYU2b260ppzYXyhpobK8u1skw3E9CCI1IZ4hdpPEUPsSQUmcVKw5PkXkYv9T1nm6kmoYEuAVqoxr3IpxoVvYA/DNKFOXxsP7BKVSk6d7NFx0DFPeKPdF4u61F/PJp004UlqDVRACklU1jvKv2YUTZVsaTZ4osi8hRHPxg5OTk5/4d31B5tC1JQ7VgAAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Max Planck Institute for Multidisciplinary Sciences\",\"correspondingAuthor\":true,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Stefan\",\"middleName\":\"\",\"lastName\":\"Glöggler\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2023-05-11 10:29:26\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-2919623/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-2919623/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1007/s00723-023-01578-z\",\"type\":\"published\",\"date\":\"2023-08-09T21:57:09+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":37102822,\"identity\":\"9b8bac01-4bc4-4886-9e0d-41ac343d3502\",\"added_by\":\"auto\",\"created_at\":\"2023-05-16 17:41:32\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":203500,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eWorkflow for the hyperpolarization of 1-\\u003csup\\u003e13\\u003c/sup\\u003eC-pyruvate with PHIP-SAH. The precursor, a phenylprogargyl ester of pyruvate is hydrogenated using pH\\u003csub\\u003e2\\u003c/sub\\u003e and a Rh-catalyst. The transfer of the polarization to the 1-13C with the MINERVA sequence is followed by a workup procedure including the addition of base to cleave the ester bond, evaporation of the acetone, addition of buffer to adjust the pH and filtration of the catalyst. The resulting aqueous solution containing the hyperpolarized 1-\\u003csup\\u003e13\\u003c/sup\\u003eC-pyruvate is injected into the tail vein of the mouse. The first injection is used to measure a series of localized spectra of the brain region, while after 10 to 25 minutes later the second injection is used to take a series of spectra from the liver region. Figure was created using Biorender and Affinity Designer 2.0\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2919623/v1/9eaf76c1e507f4867dd597c9.png\"},{\"id\":37102823,\"identity\":\"f4b9ca30-b738-4853-9e03-9b29351e43e4\",\"added_by\":\"auto\",\"created_at\":\"2023-05-16 17:41:32\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":27784,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSpectra of hyperpolarized pyruvate and reference spectrum of water measured using the 21.5 mT electromagnet. The spectrum in a) shows the \\u003csup\\u003e13\\u003c/sup\\u003eC-NMR spectrum of 40 mM hyperpolarized 1-\\u003csup\\u003e13\\u003c/sup\\u003eC-pyruvate after hydrogenation and polarization transfer acquired with one scan at 21.5 mT (Larmor frequency ~230 kHz). The spectrum b) shows the thermal \\u003csup\\u003e1\\u003c/sup\\u003eH-spectrum of 200 µL H\\u003csub\\u003e2\\u003c/sub\\u003eO acquired at 293 K with 2000 scans on the same coil and frequency of spectrum in a) at 5.69 mT. The signal of the pyruvate is enhanced by a factor of 7.75 million, which corresponds to a polarization of ~15.4 %\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2919623/v1/b525ce3a05a3fb6b8d2e270e.png\"},{\"id\":37102824,\"identity\":\"316e9df3-2d8e-46b9-9269-83401f96a6eb\",\"added_by\":\"auto\",\"created_at\":\"2023-05-16 17:41:32\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":183247,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSlice positions and plots extracted from the hyperpolarized \\u003csup\\u003e13\\u003c/sup\\u003eC-NMR spectra for one representative mouse. NMR spectra with a flip angle of 18 ° were recorded every 2 s during injection of the hyperpolarized pyruvate. From each spectrum, the pyruvate and the lactate peak were integrated and normalized to the integral of highest intensity.\\u0026nbsp; a) Position of the axial slices from which the spectra were taken on top of one slice of the sagittal proton MR image. The slice over the brain (blue) has a thickness of 18 mm, the one over the liver (red) is 21 mm thick to include the whole organ. b) Peak integrals of pyruvate (black) and lactate (blue) in the brain region plotted over time, including the fit of the lactate curve based on the AUC ratio. c) Peak integrals of pyruvate (black) and lactate (red) in the liver plotted over time, including the fit of the lactate curve based on the AUC ratio. Figure was created using Python 3.0 and Affinity Designer 2.0\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2919623/v1/f8f015b8bcbb6fd2d0cb8c6c.png\"},{\"id\":44735964,\"identity\":\"34386491-7f41-43f9-923a-8e7ec044a0ec\",\"added_by\":\"auto\",\"created_at\":\"2023-10-16 22:28:22\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":734225,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2919623/v1/36b5cd77-befa-4af1-b6ba-acf64a592309.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Hyperpolarized Multi-organ Spectroscopy of Liver and Brain using 1- 13C-Pyruvate Enhanced via Para-Hydrogen\",\"fulltext\":[{\"header\":\"1 Introduction\",\"content\":\"\\u003cp\\u003eNuclear Magnetic Resonance (NMR) Spectroscopy has a high diagnostic value and allows for the detection of metabolites and characterization of metabolism in vivo [\\u003cspan additionalcitationids=\\\"CR2 CR3\\\" citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. However, it also suffers from low sensitivity, and the tracking of metabolic kinetics in vivo is not possible with standard approaches that require a large number of averages to obtain signals above the noise level. To overcome this challenge, hyperpolarization techniques were introduced enhancing NMR signals by over four orders of magnitude [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Such large signal enhancements enable the study of metabolic real-time kinetic events in vivo. In order to do this, heteronuclei, like \\u003csup\\u003e13\\u003c/sup\\u003eC are typically used, since they have the advantage of a longer relaxation time \\u003cem\\u003eT\\u003c/em\\u003e\\u003csub\\u003e1\\u003c/sub\\u003e than protons, which allows for longer traceability of the metabolite in vivo \\u0026ndash; for \\u003csup\\u003e13\\u003c/sup\\u003eC typically up to several minutes [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eThere are different approaches to achieve hyperpolarization. Of those, dissolution Dynamic Nuclear Polarization (DNP) is the most prominent hyperpolarization technique for studying metabolism in vivo. DNP uses electron polarization to enhance the signals of close nuclear spins [\\u003cspan additionalcitationids=\\\"CR8\\\" citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. It has been used in clinical trials and produces highly polarized metabolites in high quantities [\\u003cspan additionalcitationids=\\\"CR11 CR12 CR13 CR14\\\" citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. However, it is a slow technique, taking tens of minutes to hours to produce hyperpolarized molecules, which appears to be a significant barrier to translation into clinical practice which was tried to circumvent by polarizing up to four samples at the same time [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003ePara-hydrogen (pH\\u003csub\\u003e2\\u003c/sub\\u003e) induced polarization (PHIP) is a comparably new technique in preclinical applications and offers the possibility to prepare hyperpolarized metabolites in a much shorter amount of time. In pH\\u003csub\\u003e2\\u003c/sub\\u003e nearly all hydrogen molecules occupy the same spin state, offering a high degree of spin order [\\u003cspan additionalcitationids=\\\"CR19 CR20\\\" citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. This spin order is transferred to the molecule of interest by a method, which is called Para-Hydrogen Induced Polarization by means of Side-Arm Hydrogenation (PHIP-SAH) [\\u003cspan additionalcitationids=\\\"CR23 CR24 CR25 CR26 CR27 CR28 CR29 CR30 CR31\\\" citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. The general workflow is depicted in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eA precursor, in which the molecule of interest is linked to an unsaturated side-arm via a labile bond, is hydrogenated using pH\\u003csub\\u003e2\\u003c/sub\\u003e and a catalyst in acetone. Afterwards, the spin order from the hydrogen nuclei is transferred to the \\u003csup\\u003e13\\u003c/sup\\u003eC of interest using a series of carefully timed radiofrequency pulses called the MINERVA (Maximizing Insensitive Nuclei Enhancement Reached Via para-hydrogen Amplification) sequence [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. After the transfer, the side-arm is cleaved by addition of a basic water solution. After a rapid evaporation of the acetone, the remaining aqueous solution is worked-up by addition of a buffer to adjust the pH to physiological conditions and filtration of the hydrogenation catalyst \\u0026ndash; leading to a clean solution of the hyperpolarized metabolite in water, which can then be injected into an organism. The whole procedure from the start of hydrogenation to the injection requires typically about one minute. Once injected, the hyperpolarized spin label is visible in the NMR spectra and can be used for the localization and characterization of disease or the investigation of metabolism in vivo. [\\u003cspan additionalcitationids=\\\"CR35 CR36 CR37 CR38\\\" citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e] The spectra of the metabolites can either be measured over the whole body [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e] or selectively over a region of interest, for example a single organ [\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e]. However, usually a choice has to be made as to which region should be observed, because the allowed injection volume and therefore the sensitivity of the experiment is limited especially in preclinical studies performed on rodents. Multiple injections would provide a way to work around this limitation. In vivo studies with DNP in rodents (mostly rats) demonstrated that two successive injections can be used to study the same region of interest, as to get two data points from one animal [\\u003cspan additionalcitationids=\\\"CR45\\\" citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e]. Studying large cohorts however appears to be out of reach even with multiple samples being enhanced at the same time.\\u003c/p\\u003e \\u003cp\\u003eThis work demonstrates the possibility to study two different organs of the same animal with PHIP, using two injections of hyperpolarized 1-\\u003csup\\u003e13\\u003c/sup\\u003eC-pyruvate. We recorded spectra of the region of the brain and the liver of mice within less than half an hour and analyzed them with regard to the conversion of pyruvate to lactate.\\u003c/p\\u003e \\u003cp\\u003eThis study demonstrates that the high throughput of hyperpolarized metabolites achieved with para-hydrogen enables extensive studies of complex biological systems. With this, metabolism in different organs of the same animal can be studied simultaneously, opening possibilities to study organ crosstalk and reduce the necessary number of animals by accessing more information from each one. The method is minimally invasive, the animals can be woken up again and used for further studies, enabling also longitudinal characterization of metabolism and disease progression.\\u003c/p\\u003e\"},{\"header\":\"2 Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1 In situ production of para-hydrogen\\u003c/h2\\u003e \\u003cp\\u003eMolecular hydrogen was produced by an electrolysis unit (H2PEM-510, Parker Hannifin, Cleveland, Ohio) and fed directly into a para-hydrogen generator (BPHG 90, Bruker Corporation, Ettlingen, Germany) at 36 K. The para-hydrogen was compressed to a pressure of 10 bar in a 1L buffer cylinder and led to the sample through a home-built tubing system.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2 Hyperpolarization\\u003c/h2\\u003e \\u003cp\\u003eAll chemicals except the precursor were purchased from commercial suppliers and used as received. For the samples, a solution of 13 mM Rhodium catalyst, [1,4- Bis(diphenylphosphino)butane](1,5-cyclooctadiene)rhodium(I) tetrafluoroborate, (Sigma Aldrich 79255-71-3, M\\u0026thinsp;=\\u0026thinsp;724.4 g/mol) and 95 mM of the precursor 3-(phenyl-d5)prop-2-yn-1-yl-2-13C-1,1-d2 2-oxopropanoate-1-13C in acetone-d6 was prepared. For each sample, 200 \\u0026micro;L of this solution were filled into an 8-inch 5 mm NMR tube. The hyperpolarization procedure was performed using a home-built electromagnet operating at 21.5 mT. The samples were degassed by bubbling nitrogen gas through the solution prewarmed in a water bath at 80\\u0026deg;C for about two minutes. Afterwards, the NMR tubes were placed inside a custom-built dual channel probe in the bore of the electromagnet for the PHIP experiments. A more detailed description of the system is given in [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003ePara-hydrogen was bubbled through the sample at a pressure of 7 bar for 15 seconds. Following the hydrogenation, the MINERVA pulse sequence was used to transfer the polarization from the protons to the \\u003csup\\u003e13\\u003c/sup\\u003eC of interest, in this case the 1-\\u003csup\\u003e13\\u003c/sup\\u003eC of the pyruvate moiety [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eAfter pressure release, a solution of 100 mM Na\\u003csub\\u003e2\\u003c/sub\\u003eCO\\u003csub\\u003e3\\u003c/sub\\u003e in water was added to cleave the ester bond. The acetone was removed by evaporation through flushing with nitrogen gas at a pressure of 8 bar and the pH was adjusted to physiological conditions using 1X PBS-buffered saline at pH 1.1. After passing the sample through a membrane filter with a 1.0 \\u0026micro;m pore size to remove the catalyst, the aqueous solution containing clean hyperpolarized pyruvate was ready to be injected.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3 Animal Housing and Mouse Handling\\u003c/h2\\u003e \\u003cp\\u003e Experiments were carried out in accordance with the EU directive 2010/63/EU for animal experiments and the German Animal Welfare Act (TierSchG, 2006) and were approved by the regional authorities (LANUV NRW; application number 81-02.04.2020.A157). Animals used in this study were housed under standard laboratory conditions in a 12 h light cycle with food and water available ad libitum and kept in groups of up to five. The mice were between three and four months old and fed a high-fat diet. Male mice (n\\u0026thinsp;=\\u0026thinsp;3) with a C57BL/6 N genetic background were used for the measurements.\\u003c/p\\u003e \\u003cp\\u003eThe mice were anesthetized in a dedicated container using 4% isoflurane in O\\u003csub\\u003e2\\u003c/sub\\u003e and N\\u003csub\\u003e2\\u003c/sub\\u003eO (1:3). Afterwards, the isoflurane was decreased and kept between 2% and 3% for the whole measurement. A tail vein catheter was placed and the mice were positioned head-prone on a custom-built warming water mat to ensure constant body temperature. Body temperatures as well as breathing rates were monitored during the anesthesia. The mice were positioned inside the coil alongside a urea phantom, used as a reference for the \\u003csup\\u003e13\\u003c/sup\\u003eC chemical shift scale, so that both the brain and the liver were inside the sensitive volume.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4 MR Measurements\\u003c/h2\\u003e \\u003cp\\u003eAll measurements were performed in a 9.4 T preclinical Bruker MR scanner (Bruker Corporation, Ettlingen, Germany) equipped with a 13C/1H Volume Coil by RAPID Biomedical (RAPID Biomedical GmbH, Rimpar, Germany). Localized spectroscopy of the brain region and the liver region were measured with a standard spectroscopy sequence from Bruker (NSPECT) with axial slice selection. The slices were positioned using anatomical proton images for reference, with a slice thickness for the brain of 18 mm and for the liver of 21 mm. For each measurement, a series of 128 spectra were recorded with a flip angle of 18 \\u0026deg; and TR\\u0026thinsp;=\\u0026thinsp;2 s, resulting in a measurement time of about four minutes. The acquisition was started prior to injection to record the increase of the NMR signal following pyruvate uptake in the organ of interest. The reference power for protons was adjusted automatically. The reference power for carbon was determined to be 0.19 W. The pulse duration for the spectroscopy on carbon was set to 0.2 ms. The anatomical proton images were recorded using a Bruker standard FLASH sequence with coronal and sagittal slice orientation. The number of slices was 70 with a slice thickness of 1 mm and the matrix size was 256 x 256 in a 80 mm x 80 mm FOV.\\u003c/p\\u003e \\u003cp\\u003eEach mouse was injected twice with independently generated hyperpolarized substrate. For each measurement, 2.5 \\u0026micro;L/g of body weight of pyruvate solution (40 mM) was injected through a catheter into the lateral tail vein of the mouse. The first measurement was performed on the brain, and the second on the liver. The time between the two injections was kept below half an hour. Each mouse was kept no longer than 2 hours under anesthesia. After the experiment, the mice were woken up.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5 Data Analysis\\u003c/h2\\u003e \\u003cp\\u003eThe spectra were processed using TopSpin 4.0. Phase-correction as well as line-broadening and baseline-correction were applied. All further analysis was performed using Python 3.0 in Jupyter Notebooks. In each spectrum, the peaks of pyruvate and lactate were integrated and summed up to determine the area under the curves (AUCs). From the ratio of the AUCs, the conversion coefficient \\u003cem\\u003ek\\u003c/em\\u003e\\u003csub\\u003ePL\\u003c/sub\\u003e was calculated according to [\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e], under the assumption that the back-conversion is negligible (\\u003cem\\u003ek\\u003c/em\\u003e\\u003csub\\u003eLP\\u003c/sub\\u003e=0). In order to do this, the T\\u003csub\\u003e1\\u003c/sub\\u003e of pyruvate and lactate were estimated to be T\\u003csub\\u003e1\\u003c/sub\\u003e(Pyr)\\u0026thinsp;=\\u0026thinsp;25 s, T\\u003csub\\u003e1\\u003c/sub\\u003e(Lac)\\u0026thinsp;=\\u0026thinsp;20 s.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3 Results \\u0026 Discussion\",\"content\":\"\\u003cp\\u003eFigure \\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e shows the spectrum of the \\u003csup\\u003e13\\u003c/sup\\u003eC-NMR spectrum of the hyperpolarized pyruvate, measured in the low-field spectrometer directly after para-hydrogen bubbling and application of the polarization transfer sequence. Below the pyruvate spectrum, the \\u003csup\\u003e1\\u003c/sup\\u003eH-spectrum of water, acquired at the same Larmor Frequency (~\\u0026thinsp;230 kHz) using 2000 scans is shown. Compared to thermal polarization, the signal of the pyruvate is enhanced by a factor of 7.7\\u0026nbsp;million, which corresponds to a \\u003csup\\u003e13\\u003c/sup\\u003eC polarization of 14.6% at 21.5 mT. After the full workup procedure, the concentration of the pyruvate in physiologically adjusted neat water solutions was found to be ~\\u0026thinsp;40 mM with polarization levels\\u0026thinsp;~\\u0026thinsp;6.5%. This biocompatible solution was then rapidly transferred to the MRI scanner and administered into the waiting mouse.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eEach mouse was injected twice with freshly hyperpolarized pyruvate solutions produced on time. The first injection was used to record a series of spectra of the brain, while the second one was used to record a series of spectra over the liver region. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA shows the positioning of the slices for one representative mouse. After injection, the influx of pyruvate became quickly visible in the spectra. Shortly afterward, lactate production was observed by the appearance of the corresponding signal. From the recorded spectra, the pyruvate as well as the lactate peaks were integrated and the curves plotted in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB and \\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn metabolic MR studies, the ratio of the area under the curves (AUC) for the pyruvate and the lactate peak is an important parameter frequently used to characterize metabolism. This ratio correlates with the conversion rate \\u003cem\\u003ek\\u003c/em\\u003e\\u003csub\\u003ePL\\u003c/sub\\u003e of pyruvate to lactate in the observed slice [\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e]. The AUCs were determined from the curves and the conversion rates calculated. The calculated conversion rates were then used to fit the lactate curve. The ratio of the AUCs as well as the \\u003cem\\u003ek\\u003c/em\\u003e\\u003csub\\u003ePL\\u003c/sub\\u003e in both organs including their standard deviations, were determined from the recorded spectra as summarized in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eMean and standard deviations for the ratio of the AUCs from lactate and pyruvate as well as the conversion coefficient \\u003cem\\u003ek\\u003c/em\\u003e\\u003csub\\u003ePL\\u003c/sub\\u003e determined from the spectra.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"3\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eAUC(Lac)/AUC(Pyr)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003ek\\u003c/em\\u003e\\u003csub\\u003ePL\\u003c/sub\\u003e [s\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e]\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eBrain\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0,58\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0,03\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0,044\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0,003\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eLiver\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0,38\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0,22\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0,028\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0,017\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe values suggest the tendency of a lower lactate production in the liver than in the brain. As the mice were young (3\\u0026ndash;4 months), the low lactate levels are not surprising. However, the pyruvate to lactate conversion in the brain is strongly influenced by the anesthetics, i.e. isoflurane in our case. Shortly after the initiation of the narcosis, the lactate level in the brain has been shown to stabilize, but at a significantly higher level [\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e]. This explains the elevated pyruvate to lactate conversion observed in the brain. Overall, the signal enhancement and the high throughput of the method enable subsequent injections into the same animal in a short amount of time. Sufficient signal intensities are still maintained even though the maximum possible injection volume was distributed between two injections.\\u003c/p\\u003e\"},{\"header\":\"4 Conclusions\",\"content\":\"\\u003cp\\u003eIn this work, we used two injections to successfully investigate the metabolism of two different organs in vivo using PHIP-hyperpolarized 1-\\u003csup\\u003e13\\u003c/sup\\u003eC-pyruvate. The presented results demonstrate the feasibility of this method for multi-organ studies. In particular, we believe that multiple injections have the advantage over double excitation experiments with one injection that organs can be monitored without accounting for effects due to the saturation of pulses while another organ may be investigated. Additionally, changes in pyruvate-to-lactate conversion rates are a biomarker for diagnosis, therapy and treatment response in several diseases and is hence of high interest.\\u003c/p\\u003e \\u003cp\\u003eThe presented procedure offers the possibility to investigate dependencies and cross-correlations between different regions in the body of the same animal in vivo. It paves the way for the characterization of more complex co-dependencies in disease models, also with regard to longitudinal studies. Additionally, it provides a means to reduce the number of animals used in biological studies, where often large cohorts are necessary to achieve statistical significance and makes them available by increasing the amount of available information from one animal. Additionally, it may prove advantageous to couple the presented approach with a quasi-continuous production of contrast agents [\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eWe would like to emphasize that we see the use of para-hydrogen enhanced metabolites as key to enable biological studies on large cohorts since metabolites can be rapidly generated and multiple investigations on various organs and study subjects can be carried out per day.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors thank Vera J\\u0026ouml;rke and Dr. Andreas Schmid from WSIC in T\\u0026uuml;bingen for their help with the warming mat, as well as Ulla Uhlenk\\u0026uuml;ken for the support regarding the MR scanner. Furthermore, we thank Dr. Vladimir Belov and Jan Seikowski from the synthesis facility of the MPI-NAT as well as Mario Lengauer and Christian Klaba from the workshop for fine mechanics of the MPI-NAT. S.M. thanks Dr. Lukas Kaltschnee (MPI-NAT) for useful discussions about the para-hydrogen setup. This project has received funding from the European Research Council (ERC) under the European Union\\u0026apos;s Horizon 2020 research and innovation program (Grant agreement No. 949180). S.G. acknowledges funding by the Max Planck Society and the DFG 495627437. ABS acknowledges funding support by the German Cancer Consortium (DKTK), and the DFG (#SCHM 3694/1-1, #SCHM 3694/2-1, #SFB1479).\\u003c/p\\u003e\\n\\u003cp\\u003eMaterial preparation, data collection and analysis were performed by Theresa L. K. Hune, Salvatore Mamone, In\\u0026ecirc;s Mah\\u0026uacute; and Natascha D\\u0026rsquo;Apolito. S.G. conceived the study. The first draft of the manuscript was written by Theresa L. K. Hune and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthical Approval\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eExperiments were carried out in accordance with the EU directive 2010/63/EU for animal experiments and the German Animal Welfare Act (TierSchG, 2006) and were approved by the regional authorities (LANUV NRW; application number 81-02.04.2020.A157).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no competing financial interests\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor\\u0026rsquo;s contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eMaterial preparation, data collection and analysis were performed by Theresa L. K. Hune, Salvatore Mamone, In\\u0026ecirc;s Mah\\u0026uacute; and Natascha D\\u0026rsquo;Apolito. S.G. conceived the study. The first draft of the manuscript was written by Theresa L. K. Hune and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. \\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis project has received funding from the European Research Council (ERC) under the European Union\\u0026apos;s Horizon 2020 research and innovation program (Grant agreement No. 949180). S.G. acknowledges funding by the Max Planck Society and the DFG 495627437. ABS acknowledges funding support by the German Cancer Consortium (DKTK), and the DFG (#SCHM 3694/1-1, #SCHM 3694/2-1, #SFB1479).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll data to support the findings is presented in the manuscript and is made available by the authors upon request.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eS. W. Provencher, Estimation of metabolite concentrations from localized in vivo proton NMR spectra. Magn. Reson. Med. 30, 672-679 (1993).\\u003c/li\\u003e\\n\\u003cli\\u003eM. Hajek, M. Dezortova, Introduction to clinical in vivo MR spectroscopy. Eur. J. Radiol. 67, 185-193 (2008).\\u003c/li\\u003e\\n\\u003cli\\u003eD. Bertholdo, A. Watcharakorn, M. Castillo, Brain Proton Magnetic Resonance Spectroscopy: Introduction and Overview. Neuroimaging Clin. N. 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Neuroimage, 69, 244-255 (2013).\\u003c/li\\u003e\\n\\u003cli\\u003eA. B. Schmidt, M. Zimmermann, H. de Massin, C. A. M\\u0026uuml;ller, V. Ivantaev, J. Hennig, D. v. Elverfeldt, J.-B. H\\u0026ouml;vener, Quasi-continuous production of highly hyperpolarized carbon-13 contrast agents every 15 seconds within an MRI system. Commun. Chem. 5, 21 (2022)\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"applied-magnetic-resonance\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"apmr\",\"sideBox\":\"Learn more about [Applied Magnetic Resonance](http://link.springer.com/journal/723)\",\"snPcode\":\"723\",\"submissionUrl\":\"https://submission.nature.com/new-submission/723/3\",\"title\":\"Applied Magnetic Resonance\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-2919623/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-2919623/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eHyperpolarization in nuclear magnetic resonance boosts the signals by several orders of magnitude. Using the singlet spin order of para-hydrogen to create large non-equilibrium spin polarization is a fast approach to obtain hyperpolarized metabolites in seconds. In recent years, it has attracted particular interest in the field of biomedicine because signal-enhanced and \\u003csup\\u003e13\\u003c/sup\\u003eC-enriched metabolites allow for real-time metabolic investigations in combination with imaging in vivo. With this, metabolism can be traced and characterized with spatial selectivity in the body.\\u003c/p\\u003e \\u003cp\\u003eHere, we introduce a method to use signal-enhanced metabolites to study multiple organs in separate injections to obtain real-time kinetics in vivo of these organs. Using hyperpolarized 1-\\u003csup\\u003e13\\u003c/sup\\u003eC-pyruvate, we measured the kinetics of the conversion from pyruvate to lactate in the brain and the liver of mice. This we did by injecting the hyperpolarized pyruvate two times within half an hour and using each injection to measure the spectra of one region of interest.\\u003c/p\\u003e \\u003cp\\u003eOrgan cross-talk and especially how different organs affect each other in diseases is of major interest and rarely understood, because of the high complexity of biological systems. With the proof-of-principle study provided here, we are introducing a new tool to study organ-related interaction in vivo. It allows the characterization of different organs of the same animal at the same time point, which is enabled by the fast signal enhancement achieved with para-hydrogen.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Hyperpolarized Multi-organ Spectroscopy of Liver and Brain using 1- 13C-Pyruvate Enhanced via Para-Hydrogen\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2023-05-16 17:41:27\",\"doi\":\"10.21203/rs.3.rs-2919623/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Major revision\",\"date\":\"2023-05-28T18:40:03+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2023-05-26T21:04:52+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"3ce39e91-0cca-4ea7-a6e4-d4bda9758965\",\"date\":\"2023-05-16T17:39:00+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2023-05-15T15:21:57+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2023-05-13T05:13:39+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2023-05-13T01:27:41+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Applied Magnetic Resonance\",\"date\":\"2023-05-11T10:22:33+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"applied-magnetic-resonance\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"apmr\",\"sideBox\":\"Learn more about [Applied Magnetic Resonance](http://link.springer.com/journal/723)\",\"snPcode\":\"723\",\"submissionUrl\":\"https://submission.nature.com/new-submission/723/3\",\"title\":\"Applied Magnetic Resonance\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"4e852661-053c-48d8-973b-83af314f9694\",\"owner\":[],\"postedDate\":\"May 16th, 2023\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2023-10-16T22:16:46+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-2919623\",\"link\":\"https://doi.org/10.1007/s00723-023-01578-z\",\"journal\":{\"identity\":\"applied-magnetic-resonance\",\"isVorOnly\":false,\"title\":\"Applied Magnetic Resonance\"},\"publishedOn\":\"2023-08-09 21:57:09\",\"publishedOnDateReadable\":\"August 9th, 2023\"},\"versionCreatedAt\":\"2023-05-16 17:41:27\",\"video\":\"\",\"vorDoi\":\"10.1007/s00723-023-01578-z\",\"vorDoiUrl\":\"https://doi.org/10.1007/s00723-023-01578-z\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-2919623\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-2919623\",\"identity\":\"rs-2919623\",\"version\":[\"v1\"]},\"buildId\":\"7rjqhiLT3MXkJMwkYKINL\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}