Modulation of alcohol sensitivity and alcohol tolerance by exogenous ketones in humans - 3. Abstract describing research plan for R33 The R33 phase will investigate the mechanistic effects of exogenous ketone supplementation (KS; 25g) on brain nicotinamide adenine dinucleotide (NAD+) concentrations and neurocognitive function during acute alcohol exposure in healthy adult drinkers. Building on the R21 findings demonstrating that 25g KS significantly reduced breath and blood alcohol concentrations and that 7T downfield proton magnetic resonance spectroscopy (¹H- MRS) scans reliably quantify the cerebral NAD+ peak at 9.3 ppm, the R33 will test whether ketone-induced modulation of NAD+-dependent metabolism alters central neurobiological and behavioral responses to alcohol. Forty healthy men and women (ages 21-50), who report at least one recent heavy drinking episode but do not meet criteria for alcohol use disorder, will participate in a randomized, counterbalanced, two-period crossover study. Participants will complete two experimental sessions separated by a washout period. In one session, participants will receive 25g KS; in the other, a taste-matched placebo. Thirty minutes following beverage ingestion, participants will undergo multimodal 7 Tesla MRI scanning, including structural imaging, baseline NAD+ quantification using downfield ¹H-MRS, and functional MRI during a Stop Signal Task (SST) to assess inhibitory control. Alcohol will then be administered outside the scanner to achieve a target breath alcohol concentration (BrAC) of 0.08%. Participants will return immediately to the scanner for repeated NAD+ ¹H-MRS acquisition, SST functional MRI, and exploratory spectroscopy to quantify brain alcohol and additional metabolites (e.g., β-hydroxybutyrate, glutamate). Venous blood samples will be collected to quantify alcohol, NAD+, and ketone concentrations. The primary hypothesis is that KS will increase brain NAD+ concentrations relative to placebo and attenuate alcohol-induced reductions in NAD+ levels. The primary outcome will be the interaction between treatment (KS vs placebo) and alcohol (pre- vs post-alcohol) on NAD+ concentration at the 9.3 ppm peak. Secondary hypotheses are that KS will attenuate alcohol-induced impairments in inhibitory control (indexed by stop-signal reaction time) and modulate blood oxygenation level-dependent (BOLD) activation within fronto-temporal inhibitory control networks. Exploratory analyses will examine associations between blood and brain NAD+ levels, alcohol pharmacokinetics, and cognitive performance, and will assess potential moderation by sex and genetic variation in alcohol-metabolizing enzymes. This study is innovative in combining peripheral pharmacokinetic assessment, high-field NAD+ spectroscopy, and task-based functional neuroimaging within a controlled alcohol challenge paradigm. By directly examining NAD+-dependent mechanisms in vivo, the R33 phase will provide mechanistic insight into how ketone supplementation may alter alcohol metabolism and sensitivity at both peripheral and central levels. Findings will advance understanding of cellular and molecular mechanisms of alcohol tolerance and may identify NAD+- dependent metabolic modulation as a novel target for prevention or intervention strategies for alcohol misuse and alcohol use disorder. 4. Aims page revision The Specific Aims have been modified in a minor way from the original peer-reviewed and approved application. In the originally proposed R33 phase, participants were to receive either 10g or 25g of ketone supplement (KS), with final dose selection contingent upon R21 findings. Based on the R21 results, which demonstrated that 25g KS significantly reduced breath and blood alcohol concentrations (p<0.05) whereas 10g did not produce significant pharmacokinetic effects, the R33 phase will utilize the 25 g dose exclusively. This modification reflects data-driven optimization consistent with the original study design and milestone framework. The overall scientific premise, hypotheses, and mechanistic focus on
