The Effect of Ketone Bodies and Lipid Nanoparticles on Lethal Infection

Authors:
Cuthbert Simpkins, Gelilla Daniel, Gelilla Daniel, Vidit Minda, William Gutheil

Body of Abstract:
Introduction: With the rise of multidrug resistant organisms, sepsis has become increasingly difficult to treat. The discovery of a treatment that is not solely reliant on standard antibiotics is vital. We decided on an approach that optimized macrophage activity. Studies have shown that the ketone, beta-hydroxybutyrate, suppresses differentiation of pro-inflammatory M1 macrophages and augments anti-inflammatory M2 macrophage lineages. Our previous studies demonstrated that lipid nanoparticles (VBI-1) reduce the expression of M1 macrophage genes. Hence, we tested the efficacy of ketones [Ethyl Acetoacetate (EAA), Beta-Hydroxybutyrate (BHB), and Ethyl Hydroxybutyrate (EHB)] and VBI-1 on the survival of rats subjected to lethal intra-abdominal infection.

Methods: We utilized a cecal ligation and laceration (CLL) model to induce lethal sepsis in adult male and female Sprague-Dawley rats. The cecum was ligated below the ileocecal junction with silk suture; then, a 0.5 cm laceration was made in the distal cecum. The cecum was then returned to the peritoneal cavity and the laparotomy was closed. The control rats received a intraperitoneal (IP) injection of isotonic aqueous solution in a volume equal to 8% of the rat’s body weight. The treatment group received an IP injection of the same solution with 125 mM of the ketones (EAA, BHB, EHB), VBI-1, or a combination of VBI-1 and ketones. The injection was delivered thirty minutes or five hours after the procedure. No source control, antibiotics, or intravascular fluids were given. Survival of rats beyond 24 hours was considered a success. Rats were observed for a maximum of thirty days. Statistical analysis was carried out using a one-way ANOVA and LSD post-hoc test.

Results: All control rats died within 24 hours of the CLL. Within thirty minutes post-CLL, there was no statistical significance between rats that received EAA and control (p=0.072). 60% survival was noted with BHB (p=0.009). Survival of 80% was shown with EHB (p=0.001). We further assessed the efficacy of EHB by performing the injection five hours post-CLL with a survival rate of 67% (p = 0.004). 40% survival was noted with VBI-1 alone (p=0.029). We then injected a combination of VBI-1 and EHB thirty minutes post-CLL which showed a survival of 100% of rats (p <0.0001). We further analyzed the combination at five hours post-CLL with a survival of 50% (p=0.015). To better understand the mechanism, we performed an in vitro study to evaluate the minimum inhibitory concentrations of EHB and BHB against E. coli and MRSA. The EHB exhibited the greatest level of bacterial suppression, most effectively against E. coli. Conclusion: EHB is a ketone with minimal research on its role in the mitigation of sepsis. It exhibits an improvement on survival with and without VBI-1 in a lethal model of sepsis without reliance on standard antibiotics. Continued research is being conducted to further understand its mechanism of action in macrophage activity.