The aim was to study the influence of isocitrate dehydrogenase 1 mutation IDH1R132H on α-ketoglutarate metabolism in an acute myeloid leukemia cell line in vitro using hyperpolarized [1-¹³C]α-ketoglutarate and ¹³C metabolic magnetic resonance spectroscopy (¹³CMMRS).The isocitrate dehydrogenase 1 (IDH1) is an intracellular enzyme, located in the cytosol, which normally catalyzes the oxidation of isocitrate to α-ketoglutarate (αKG). However, in several cancer types, such as glioma or acute myeloid leukemia (AML), R132H mutation in the IDH1 gene causes a functional change, which plays a significant role in further cancer development^1,2. The monoallelic mutation causes a gain of function, resulting in an alternative product from αKG’s oxidation; the oncometabolite 2-hydroxyglutarate (2-HG)³. This causes a 10–100 times higher accumulation of 2-HG in the cancerous than in healthy tissue^4,5, allowing even its detection by ¹H magnetic resonance spectroscopy⁶. Among other alternations caused by IDH1 mutation, expression of branched chain amino-acid transaminase 1 (BCAT1) is reduced causing decrease in αKG to glutamic acid (Glu) metabolic pathway^7,8. See Fig.1 for detailed metabolic pathways description. Interestingly, this mutated version of the IDH1 gene, was discovered in 8% of all acute myeloid leukemia (AML) genomes tested and was largely connected with normal cytogenetic status; it was present in 16% cytogenetically normal samples⁹. Dissolution dynamic nuclear polarization (dDNP) of ¹³C-labeled compounds enhances the NMR signal in vivo by over 10,000 times, which enables studying metabolic pathways in real time¹⁰.An acute myeloid leukemia (AML) cell line, carrying IDH1R132H, Npm1cA and NrasG12D was derived from mutant IDH1R132H (Cre recombined) mouse. As a control AML cell line carrying Npm1cA and NrasG12D only was used. Both cell lines were cultured in X-Vivo 20 medium (Lonza, Basel, Switzerland) supplemented with IL-3, IL-6, SCF (Peprotech, Rocky Hill, NJ, USA) and 5% FCS (Biochrom, Berlin, Germany) and grown on special low-attachment plates (Corning, Corning, NY, USA). Cells were kept in a humidified atmosphere at 5% CO₂ and 37°C. Mixture of [1-¹³C]α-ketoglutaric acid (CIL, Massachusetts, USA), 20 mM trityl radical OX063 (GE Healthcare, Amersham, UK), and 0.6 mM Dotarem^® (Guerbet, Roissy, France) in DMSO was hyperpolarized using Hypersense™ polarizer (Oxford Instruments, Abingdon, UK) at ~94 GHz. The sample was dissolved in PBS buffer to 20 mM at pH 7.4. The solution was mixed with AML-IDH1mut/IDH1wt cell suspension (5×10⁷ cells/mL) resulting in 5 mM final [1-¹³C]α-ketoglutarate concentration. The mixture was transferred to 5mm NMR tube and measured at 1 Tesla Spinsolve^® NMR spectrometer (Magritek, San Diego, USA) using FA = 10°, TR = 3 s, NS = 100. We were able to observe a differences in the metabolism of [1-¹³C]α-KG caused by the IDH1R132H mutation using hyperpolarized [1-¹³C]α-KG and metabolic magnetic resonance spectroscopy (MMRS) in AML cell line in vitro. (Fig.2) The longitudinal (or spin-lattice) relaxation time (T₁) allowed long enough measuring time to obtain the metabolism with T₁ = 57 ± 3 s for [1-¹³C]α-KG and 56 ± 3 s for [1-¹³C]α-ketoglutarate hydrate at 1 Tesla in vitro (n=4). Our data proved that IDH1R132H mutation causes a significant decrease of αKG (172.6 ppm) to Glu (177.2 ppm) metabolism by 89 ± 8% (Fig.3). Therefore, we assume that expression of BCAT1 is reduced in AML as in previously reported gliomas.^7,8 Due to a low spectral resolution of 1 Tesla spectrometer, we are not absolutely positive, whether the peak at 183.7 ppm belongs to oncometabolite 2-HG or to [5-¹³C]αKG, even though 99% [1-¹³C]αKG was used for the experiments.