László G. Boros 1,2,3,* & Gábor Somlyai 4,*

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1 doi: /HYDLLCHU2014DDWCONFKEYNOTE Deuterium depletion simulates mitochondrial matrix metabolic water use via NADPHdependent reductive synthesis by fumarate hydratase, oxidative pentose cycling and the SOGC-pathway László G. Boros 1,2,3,* & Gábor Somlyai 4,* 1Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, CA 90502, USA.; 2 SIDMAP, LLC, Los Angeles, CA 90064, USA.; 3 Los Angeles Biomedical Research Institute, LABIOMED, Torrance, CA 90502, USA.; 4 HYD, LLC for Cancer Research & Drug Development, Budapest, H-1124, Hungary, EU. A key metabolic event during megaloblastic hemopoesis [1] and eukaryotic cell transformation by either Nrf2 [2] or PGC-1α [3] is the activation of irreversible direct glucose oxidation reactions in the pentose cycle to maintain NADPH-dependent reductive nucleotide synthesis and membrane lipogenesis. Clear cell kidney tumors become exclusively extracellular free water dependent due to their mitochondrial fumarate hydratase mutation, which results in the consumption of pentose cycle and cytoplasmic fumarate hydratase derived NADPH for reductive fatty acid synthesis and ketoglutarate( ) carboxylation [4, 5]. The possible role of the increased heavy hydrogen content of cytoplasmic free water, deuterium, in cell cycle regulation and the antiproliferative effect of deuterium depletion have been reported [6]. We herein demonstrate that deuterium depletion in extracellular (free) water decreases nucleotide and nuclear membrane behenic- and lignoceric acid synthesis, which recapitulates restored mitochondrial fumarate hydratase catalytic function in kidney cells for NADPH production. Targeted [1,2-13 C 2 ]-D-glucose to [1-13 C 1 ]-D-ribose and 13 C- glutamate fate associations indicate that the pentose cycle (oxidative branch), cytoplasmic fumarate hydratase and the serine synthesis, one-carbon (folate) metabolism with the glycine cleavage system (SOG pathway) [7] concomitantly mediate low-deuterium reductive NADPH-dependent macromolecule synthesis. We conclude that impaired mitochondrial proton transport forces the TCA cycle to branch at fumarate and succinate, which limits the low natural deuterium containing fatty acid [8] oxidation product, metabolic water [9], to enter metabolism. Taking proton transfer from food and free water for NADPH production over by cytoplasmic fumarate hydratase, the oxidative branch of the pentose cycle [10-12] and the SOGC pathway opens new windows for deuterium depletion to normalize cellular functions [13-16], after precise individual cellular metabolic profiles are obtained [17]. We review support from synergistic dietary high fat ketogenic (KD) and hyperbaric oxygen treatment (HBO₂T) with significant anti-tumor effects in models of systemic metastatic cancer, which directly point to the fundamental involvement of complex-iv and its deuterium depleted matrix water from dietary fatty acid oxidation in suppressing tumor progression [18]. Metabolic adaptation of immune modulator synthesis to TCA cycle branching at succinate [19] deprives metabolic water production, especially when it is accompanied by intense ATP synthesis [20]. These processes are explored as therapeutic targets for immune disorders, where deuterium depletion plays a well-defined role [21].

2 Fig. 1. Glucose and water with 150 ppm and fatty acids with a lower average 118 ppm deuterium load (in water equivalents) pass either hydrogen or deuterium to NADPH, which is the reducing substrate for all macromolecule DNA and fatty acid synthesis reactions in eukaryote cells. a) The Krebs-Szent-Györgyi (tricarboxylic/citric acid - TCA) cycle produces citrate from both fatty acids and carbohydrate derived acetyl-coa, but citrate synthase (CS), aconitase and fumarate hydratase replace deuterium in all matrix intermediates using matrix water s hydrogen during hydrolysis. Matrix/metabolic water has a lower, fatty acid matching deuterium content due to cytochrome c oxidase action, which transfers the hydrogen of fatty acids to oxygen. Saturated fatty acids have decreased natural deuterium content due to plant enzyme discrimination against deuterium during photosynthesis. In healthy cells matrix water hydrogen saturates NADPH via malate, b) citrate and isocitrate shuttling, followed by NADP+-dependent irreversible cytoplasmic isocitrate dehydrogenase action. c) On the other hand, peroxisomal and pentose cycle maintain a NADPH pool with a deuterium content matching carbohydrate and free (drinking) water derived deuterium. d) Nevertheless, normal cells with active mitochondria produce lighter NADPH, thus DNA and nuclear membrane structural and functional hydrogen bonds remain stable, by the robust cytoplasmic isocitrate dehydrogenase flux of the resting cycle phase. (Modified with Permissions Europe/NL Permissions.Dordrecht@springer.com; TN Use of Open Access article figure)

3 Fig. 2. Decreased mitochondrial function due to the activation of diverse oncogenic signals, hypoxia, lactic acidosis, fumarate hydratase and/or isocitrate dehydrogenase mutations not only diminish a) the TCA cycle and matrix water use for b) citrate/isocitrate shuttling, but reverse these processes for reductive carboxylation that depend on c) peroxisomal, pentose cycle and SOG-pathway (not shown here) metabolism, which take over NADPH production with a deuterium content matching that of higher carbohydrate and free water deuterium contents. d) Therefore, cell transformation and diminished mitochondrial function add heavier and less stable NADPH, DNA, nuclear membrane structural and functional hydrogen pool primarily via pentose cycle and SOG-pathway metabolism. Such metabolic switch sets the stage, along with growth signaling, for aneuploidy and long-chain saturated nuclear fatty acid products (lignocerate and behenate) necessary for increased nuclear membrane/cytoplasmic ratios; all hallmarks of cancer with severely altered nuclear functions and frequently repeated mitosis. (Modified with Permissions Europe/NL Permissions.Dordrecht@springer.com; TN Use of Open Access article figure)

4 Fig. 3. Decreasing deuterium content in drinking water to e.g.: 100 ppm, which is closer to the average 118 ppm deuterium load (in water equivalents) of natural and dietary fatty acids, reprograms cellular metabolism even when a) the Krebs-Szent-Györgyi (tricarboxylic/citric acid TCA) cycle is still insufficient to produce low deuterium citrate, isocitrate and malate with metabolic water. Matching matrix/metabolic water deuterium depletion in drinking water, even without b) citrate/isocitrate shuttling and fumarase and/or isocitrate dehydrogenase action, c) peroxisomal, pentose cycle and SOG-pathway metabolism, alike, will maintain a NADPH pool with low deuterium content. d) In turn, low deuterium containing water simulates metabolic events described for restoring fumarate hydratase function with more balanced, normal cell-like mitochondria that produce lighter NADPH, DNA and nuclear membrane structural and functional hydrogen bonds, which prevent or even reverse the long lasting metabolic effects of oncogenic signals to produce aneuploidy with nuclear membrane malfunctions. (Modified with Permissions Europe/NL Permissions.Dordrecht@springer.com; TN Use of Open Access article figure)

5 Fig. 4. Normal mitochondria balance ATP synthesis with water production evenly by distributing protons between the inter-membrane space for ATP synthesis, and the cytochrome-c/complex-iv proteins for water production. Cytochromec oxidize produces low deuterium metabolic water from saturated fatty acid derived protons, which help recycling fumarate and succinate via fumarate hydratase as malate (green arrows) in the matrix of normal mitochondria. Allergen/antigen and hormone activated immune cells present with a branching TCA cycle (red arrows) at succinate and fumarate, which depletes complex-iv of protons, thus limiting metabolic water production (red crosses). Mitochondria with branching, due to hypoxia, succinate dehydrogenase, fumarate hydratase mutations and with increased proton channeling towards ATP synthase, must redirect the hydration of fumarate to the cytoplasm, using free, high-deuterium containing extracellular/cytoplasmic water. Therefore, deuterium depleted water in cells with defective mitochondria limits deuterium load and regulates prostaglandin and inflammatory mediator production, nuclear membrane stability, cellular expansion, aneuploidy, as well as immune- and tumorigenic signals (Patents WO US [PCT/HU2010/000044] Compositions comprising water with deuterium for the prevention or treatment of allergic diseases and a process for the preparation thereof -

6 References: [1] Boros, LG, Steinkamp, MP, Fleming, JC, Lee, WN, Cascante, M, Neufeld, EJ. Defective RNA ribose synthesis in fibroblasts from patients with thiamine-responsive megaloblastic anemia (TRMA). Blood 102, (2003). Doi: /blood [2] Singh, A. et al. Transcription factor NRF2 regulates mir-1 and mir-206 to drive tumorigenesis. J Clin Invest 123, (2013). doi: /JCI66353 [3] Bhalla, K. et al. PGC1α promotes tumor growth by inducing gene expression programs supporting lipogenesis. Cancer Res 71, (2011). doi: / CAN [4] Mullen, A.R., Wheaton, W.W., Jin, E.S., Chen, P.H., Sullivan, L.B., Cheng, T., Yang, Y., Linehan, W.M., Chandel, N.S., DeBerardinis, R.J. Reductive carboxylation supports growth in tumour cells with defective mitochondria. Nature 481, (2011). [5] Yang, Y., et al. Metabolic reprogramming for producing energy and reducing power in fumarate hydratase null cells from hereditary leiomyomatosis renal cell carcinoma. PLoS ONE 8, e72179 (2013). doi: /journal.pone [6] Somlyai, G., et al. Naturally occurring deuterium is essential for the normal growth rate of cells. FEBS Lett 8, 1-4 (1993). [7] Tedeschi, P.M., et al. Contribution of serine, folate and glycine metabolism to the ATP, NADPH and purine requirements of cancer cells. Cell Death Dis 4, e877 (2013). doi: /cddis [8] Billault, I., Guiet, S., Mabon, F., Robins, R. Natural deuterium distribution in long-chain fatty acids is nonstatistical: a site-specific study by quantitative 2H NMR spectroscopy. Chembiochem 2, (2001). [9] Vander Heiden, MG. Targeting cancer metabolism: a therapeutic window opens. Nat Rev Drug Discov 10, (2011). doi: /nrd3504 [10] Mellanby K. Metabolic water and desiccation. Nature 150, (1942). doi: /150021a0 [11] Boros, L.G., et al. Nonoxidative pentose phosphate pathways and their direct role in ribose synthesis in tumors: is cancer a disease of cellular glucose metabolism? Med Hypotheses 50, (1998). [12] Boros, L.G., Lee, W.N., Cascante, M. Imatinib and chronic-phase leukemias. N Engl J Med 347, (2002). doi: /NEJM [13] Gyöngyi, Z., et al. Deuterium depleted water effects on survival of lung cancer patients and expression of Kras, Bcl2, and Myc genes in mouse lung. Nutr Cancer 65, (2013). doi: /

7 [14] Krempels, K., Somlyai, I., Somlyai, G. A retrospective evaluation of the effects of deuterium depleted water consumption on 4 patients with brain metastases from lung cancer. Integr Cancer Ther 7, (2008). doi: / [15] Gyöngyi, Z., Somlyai, G. Deuterium depletion can decrease the expression of C-myc Ha-ras and p53 gene in carcinogen-treated mice. In Vivo 14, (2000). [16] Krempels, K. et al. A retrospective study of survival in breast cancer patients undergoing deuterium depletion in addition to conventional therapies. J Cancer Res Ther 1, (2013). [17] Metallo, C.M., Walther, J.L., Stephanopoulos, G. Evaluation of 13C isotopic tracers for metabolic flux analysis in mammalian cells. J Biotechnol 144, (2009). doi: /j.jbiotec [18] Poff, A.M., Ari, C., Seyfried, T.N., D'Agostino, D.P. The ketogenic diet and hyperbaric oxygen therapy prolong survival in mice with systemic metastatic cancer. PLoS One. 8: e65522 (2013). doi: /journal.pone [19] Tannahill, G.M., et al. Succinate is an inflammatory signal that induces IL-1β through HIF-1α. Nature 496(7444), (2013). doi: /nature11986 [20] Gatza, E., Wahl, D.R., Opipari, A.W., Sundberg, T.B., Reddy, P., Liu, C., Glick, G.D., Ferrara, J.L. Manipulating the bioenergetics of alloreactive T cells causes their selective apoptosis and arrests graft-versus-host disease. Sci Transl Med 3(67), 67ra8. doi: /scitranslmed [21] Somlyai, G. Defeating Cancer! The Biological Effect of Deuterium Depletion. ISBN: Type: B/W

CITRIC ACID CYCLE ERT106 BIOCHEMISTRY SEM /19 BY: MOHAMAD FAHRURRAZI TOMPANG

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