“What to eat or what not to eat—that is still the question” - Reply

We appreciate the interest of Drs Boros, Collins, and Somlyai in our recent publication on ketone body metabolism in rat glioma models. The authors address 2 issues in their letter: (i) the low degree or lack of ketone body oxidation in RG2 and 9L cells in vitro, which contrasted the in vivo data of RG2 and 9L rat brain tumors, and (ii) the lack of an inhibiting effect of the ketogenic diet on tumor growth.

The authors point out that the cells were not conditioned to the presence of ketone bodies, as was the case during the actual experiment in which ¹³C-labeled [2,4-¹³C₂]-beta-hydroxybutyrate (BHB) was added to the medium. We already addressed this in our research and performed the experiments Boros et al suggest we should have done. The results were presented in the original paper in the section “Effect of Ketogenic Diet-Mimicking Cell Culture Conditions, and Explanted 9L Cells.”¹ Nutrient conditioning had no effect on in vitro ketolysis in 9L and RG2 cells.

Boros et al point out that the high ketogenic amino acid content in the medium would hamper the uptake of additional ketone substrates. There are a number of amino acids in the medium (0.8 mM) that could be metabolized to ketoacids. Yet this requires transaminases to be present in the medium. In addition, the RG2 cells did show some degree of ketone body oxidation (Fig. S1 in original paper),¹ indicating that any monocarboxylate transporter 1 inhibition was insufficient to block BHB uptake in RG2 cells.

Next Boros et al address the admittedly challenging question of why we did not observe a slowing down of tumor growth in RG2- and 9L-bearing rats, while most other studies do report such therapeutic effect in glioma-bearing mice. Their mechanistic explanation relies on the assumption that the ketogenic diet used in our study has a high level of deuterium that “strongly influences the therapeutic value of ketogenic diets through reducing intracellular compartmentalized water with particular deuterium loads, whereby deuterons pose significant kinetic isotope effects.” The reference accompanying this statement is a non–peer reviewed online commentary. The natural abundance of ²H on earth is 0.011%. Even an unrealistic 100-fold increase in natural abundance ²H would result in only 1% of protons in fatty acids from the ketogenic diet to be replaced by ²H. The kinetic isotope effect (KIE) as referred to by Boros et al would reduce rates of enzymatic reactions breaking carbon-deuterium bonds. As such, the presumed high deuterium content of our ketogenic diet as suggested by Boros et al would slow down ketone body metabolism via KIEs on ketolytic enzymes yet also produce deuterated metabolic water. The extent of ketone body metabolism is exactly what we measured in vivo with ¹³C MR spectroscopy. In contrast to what Boros et al suggest, the relative rate of ketone body oxidation was similar between tumorous and nontumorous brain tissue, and was increased when animals were put on the ketogenic diet (Supplementary Table 2).¹ Interestingly, if the level of the elusive deuterated water was indeed high, if anything it could have had a tumor growth–inhibiting effect, as observed by Rodrigues et al after providing 50% (v/v) ²H₂O for 9 days in glioma-bearing rats (which is several orders of magnitude above the ²H enrichment anticipated from a diet with slightly increased ²H content).²

Funding

H. M. De Feyter was supported by fellowship 10A087 from the American Institute for Cancer Research, and a 2013 Discovery Award from the American Brain Tumor Association. As a member of the Yale Cancer Center, H. M. De Feyter is supported by NIH grant CA-16359 from the National Cancer Institute.