Ketone Ester and Salt (KEAS) in Young Adults

Excessive salt consumption is widespread across the United States and remains a leading risk factor for developing hypertension and cardiovascular disease (CVD). What has been less appreciated until recently is that high salt (HS) plays a large role in the development of chronic inflammation, which importantly, plays a critical role in the development of CVD. The well-documented relation between HS, hypertension, and CVD risk along with the ubiquitous HS intake in the United States demonstrate a critical need for investigation into mechanisms of salt-induced CVD; and the development of therapeutic strategies to combat the consequences of HS, particularly in at-risk populations. The investigators have identified the liver-derived ketone body β-hydroxybutyrate (β-OHB) as a potential target to combat the negative cardiovascular health effects of HS. Circulating β-OHB concentration typically increases in response to endurance exercise or calorie restriction, both of which also reduce blood pressure (BP) and lower CVD risk. Further, recent data suggest that increasing circulating β-OHB concentrations, using short-term exogenous ketone supplements, also improves resting BP and vascular function in humans. Interestingly, chronic HS consumption suppressed endogenous hepatic β-OHB production in rats, but nutritionally upregulated hepatic β-OHB production attenuated the adverse effects of HS in the rats. Specifically, using 1,3-butanediol to increase β-OHB counteracts the adverse effects of HS on resting BP, in part by acting as a vasodilator, and attenuating inflammation. Our human pilot data also indicates that HS suppresses circulating β-OHB concentration in healthy young adults. However, there is a knowledge gap regarding whether increasing β-OHB during HS intake can counteract the negative effects of HS on BP and cardiovascular function in humans. Therefore, the investigators will measure resting blood pressure, endothelial function, kidney blood flow, BP responses during and after submaximal aerobic exercise and inflammatory markers in blood and isolated immune cells (i.e., monocytes). Recognizing that HS does not increase BP in everyone, several studies consistently indicate that short-term HS ingestion (days to weeks) leads to endothelial dysfunction and exaggerated BP reactivity during submaximal exercise in rodents and humans. Importantly, endothelial dysfunction contributes to atherosclerotic cardiovascular disease. Additionally, exaggerated BP responses during aerobic exercise (i.e., BP reactivity) have prognostic value for future hypertension, coronary disease risk, and cardiovascular mortality. Apart from leading to exaggerated exercise BP reactivity, the investigators have found that HS also reduces the magnitude of post-exercise hypotension (PEH) after an acute bout of submaximal aerobic exercise in healthy adults. Importantly, the reductions in BP observed after a single bout of exercise are associated with longer-term exercise reductions in BP, suggesting that some of the benefits of aerobic exercise on BP status are the result of transient reductions in BP resulting from an acute bout of exercise. Regarding the effects of HS on the immune system and inflammation, microenvironments with elevated concentrations of sodium increase the prevalence of proinflammatory phenotypes within specific immune cell subsets. For example, HS conditions activate monocytes to produce pro-inflammatory cytokines. Thus, HS-induced immune system dysregulation may further amplify BP dysregulation and CVD risk. The investigators hypothesize that increasing circulating β-OHB concentration via ketone supplementation will counteract the negative effects of HS on these measures of cardiovascular health. Interestingly, elevating β-OHB leads to greater sodium excretion under HS conditions (indicative of restoration of plasma volume homeostasis) and restores nitric oxide-dependent vasodilation in rodents. Thus, the investigators hypothesize that ketone supplementation will improve endothelial function and BP regulation during and after exercise. Though exploratory, the investigators hypothesize that β-OHB supplementation blunts the HS-induced proinflammatory alterations in monocytes and blood samples using parallel in vitro and applied approaches.

Participants will report to the laboratory for four visits. At the first visit, consent for study participation will be obtained and participants will be screened for eligibility. Participants will then be randomly assigned to a crossover schedule for exposure to salt and ketone supplementation. Supplementation conditions include \[A\] Placebo capsules and Placebo beverage, \[B\] Salt capsules and Placebo beverage, and \[C\] Salt capsules and Ketone beverage. Each participant will be exposed to all three conditions, however, the order of exposure will be randomly assigned. Participants will consume their placebo/salt capsules three times per day and their placebo/ketone beverage three times per day.

Participants will consume the first assigned supplement combination for nine days prior to their first scheduled experiment visit (i.e., first experimental visit is day 10 of supplement combination#1). After a washout period, participants will consume the next randomly assigned supplement combination for nine days prior to the second scheduled experiment visit (i.e., day 10 of supplement combination #2). After another washout period, participants will consume the final randomly assigned supplement combination for nine days prior to the third scheduled experiment visit (i.e., day 10 of supplement combination #3). Participation will end after the third experimental visit has been completed.

• Flow mediated dilation (FMD) [Time frame: This measure is completed on day 10 of each 10-day intervention (low salt, high salt, high salt+ ketone) over 3-4 months and values will be compared across interventions.]
• Post-exercise resting blood pressure [Time frame: This measure is completed on day 10 of each 10-day intervention (low salt, high salt, high salt+ ketone) over 3-4 months and values will be compared across interventions.]
• Post-exercise ambulatory blood pressure [Time frame: This measure is completed on day 10 of each 10-day intervention (low salt, high salt, high salt+ ketone) over 3-4 months and values will be compared across interventions.]
• Kidney blood velocity [Time frame: This measure is completed on day 10 of each 10-day intervention (low salt, high salt, high salt + ketone) over 3-4 months and values will be compared across interventions.]
• Passive Leg movement [Time frame: This measure is completed on day 10 of each 10-day intervention (low salt, high salt, high salt+ ketone) over 3-4 months and values will be compared across interventions.]