**3. Metabolism of Ketones: Ketogenesis and Ketolysis**

The human body produces energy as ATP generated by the mitochondria, for survival. The main energy sources are carbohydrates, fats, amino acids (predominantly glutamine), lactate, and ketone bodies (Figure 1).

Ketone bodies, that are produced during ketogenesis are of three types: acetone, acetoacetate (AcAc), and 3-hydroxybutyrate (BHB). The mainly produced ketone body molecule during ketogenesis is BHB, mainly the R-BHB enantiomer. BHB levels increase in plasma much faster than acetoacetate or acetone, for example, in prolonged fasting [19]. During the conditions such as fasting or vigorous exercise, the consumption of blood glucose lowers insulin levels, turning on ketogenesis, and triglycerides are catabolized to fatty acids that are converted into ketone bodies in the liver [2]. Ketone bodies arrive in metabolically active tissues (muscle, brain) via the blood stream to be metabolized into acetyl-CoA and eventually ATP (Figure 1). BHB is converted, in extrahepatic tissues, by the enzyme 3-hydroxybutyrate dehydrogenase (BDH1) to AcAc [2], which generates Acetyl-CoA [20,21] by exchanging the CoA-fraction from succinyl-CoA [22] by succinyl CoA-oxoacid transferase (SCOT) [23]. Acetyl-CoA enters the TCA cycle and produces

22 ATP per molecule post oxidative phosphorylation. Acetoacetate can be converted also to 3-hydroxybutyrate by BDH or to acetone via a non-enzymatic decarboxylation [20].

**Figure 1.** Schematic Diagram of the metabolic pathways of key energy sources in the human body. NAD: nicotinamide adenine dinucleotide; FADH: reduced flavin adenine dinucleotide; NADH: nicotinamide adenine dinucleotide (NAD) + hydrogen (H); PDH: pyruvate dehydrogenase; LDH: lactate dehydrogenase; CPT: carnitine palmitoyl transferase; ß HOB: beta hydroxybutyrate; BDH: D-3-hydroxybutyrate dehydrogenase; SCOT: succinyl-CoA acetoacetate transferase; AcAc: acetoacetate; CoA: coenzyme A; CoA-SH: coenzyme A with sulfhydryl functional group; CO2: carbon dioxide; H2O: water; ADP: adenosine diphosphate; ATP: adenosine triphosphate; Pi: phosphorylated forms of phosphatidylinositol; NH3: ammonia; NH4: ammonium; H+: hydrogen ion; HMG-CoA: ß-hydroxy-ß-methylglutaryl-CoA; MCT: monocarboxylate transporters; GLUT: glucose transporter; AAT: amino acid transporter; ALT: alanine amino transferase; PC: pyruvate carboxylase; MPC: mitochondrial pyruvate carrier.

The short-chain fatty acids (SCFAs), butyrate, acetate, and propionate (in a molar ratio of 3:1:1), are produced as microbial fermentative end-products of undigested/unabsorbed dietary carbohydrates [24,25]. Butyrate is produced by acetate and/or lactate-utilizing butyrate-producing bacteria through the butyryl-CoA: acetate CoA-transferase pathway [24,26,27]. This pathway is typically present in Firmicutes, within Lachnospiraceae (*Eubacterium hallii*, *Eubacterium rectale*, *Coprococcuscatus*, *Roseburia intestinalis*), Ruminococaceae (*Faecalibacterium prausnitzii*), and *Clostridium* spp. in which butyrate and acetyl-CoA are formed from butyryl-CoA and the transformation of the CoA moiety to the external acetate molecule [25–29], while *Bifidobacterium* and *Lactobacillus* spp. use lactate to produce SCFAs. These molecules, similarly, and in combination with ketone bodies, have an anti-inflammatory role via an epigenetic mechanism such as butyrate-associated HDAC inhibition [30].

#### **4. Endogenous Sources of Ketone Bodies**

Ketone bodies might derive from endogenous or exogenous sources (Figure 2): endogenous ketones are normally present in our bloodstream and are produced mostly by the liver and by certain species of gut bacteria.

**Figure 2.** Endogenous and exogenous metabolic pathways. NAD: nicotinamide adenine dinucleotide; FADH: reduced flavin adenine dinucleotide; NADH: nicotinamide adenine dinucleotide (NAD) + hydrogen (H); CPT: carnitine palmitoyl transferase; ß HOB: beta hydroxybutyrate; BDH: D-3-hydroxybutyrate dehydrogenase; SCOT: succinyl-CoA acetoacetate transferase; AcAc: acetoacetate; CoA: coenzyme A; ADP: adenosine diphosphate; ATP: adenosine triphosphate; Pi: phosphorylated forms of phosphatidylinositol; H+: hydrogen ion; HMG-CoA: ß-hydroxy-ß-methylglutaryl-CoA; MCT: monocarboxylate transporters.

Microbiota-derived SCFAs, primarily butyrate, acetate, and propionate are metabolites produced by gut microbiota via dietary non-digestible carbohydrate fermentation [24,25]. SCFAs play a significant role in CHO and lipid metabolism. Butyrate and acetate are used as precursors for lipid synthesis (cholesterol, long-chain fatty acids), whereas propionate is used as a precursor for hepatic gluconeogenesis [24,31]. SCFAs are influenced by the diet (fiber, fats, plant-based proteins) and are important during pregnancy and lactation, controlling the formation of infant gut microbiota. The difference in SCFA-producing bacteria in gut microbiota leads to a different anti-inflammatory state with an impact on inflammatory conditions (e.g., obesity, asthma) [11,12].

In physiology, ketosis can be achieved by fasting, via exogenous supplementation, or the consumption of a ketogenic diet. Fasting from 12–16 h up to 48 h increases serum

ketone body concentrations but may elicit inconsistent effects on performance in humans and animal models [32].

#### **5. Exogenous Sources of Ketone Bodies**

Exogenous ketone bodies are able to obtain ketosis [1]. Exogenous ketone bodies can be acquired from diet or supplements, such as medium-chain triglyceride (MTC) oils, ketone salts, or esters. MTCs are usually sold as oils or as lyophilized powders and are used to provide the mitochondria with non-carbohydrate energy. They are composed of 8–10 carbon fatty acid chains and they are capable of inducing ketosis thanks to the excess of acetyl CoA produced by the liver while it metabolizes MTC. Ketone salts can be a good alternative to ketogenic diets thanks to their application versatility. They are easy to take and can quickly raise the blood level of ketones. These compounds have a low risk of health issues in humans, and the only concern happens because of the sodium salts as they could increase blood-free sodium levels [33]. Another issue could be the racemic mixture that is present in this product; usually, BHB salts are a combination of D-BHB and L-BHB. It is known that the L form cannot be metabolized by the liver and remains in the blood until it is eliminated through urine or feces. Ketone esters are produced by synthesis, linking one molecule of BHB. Ketone esters are more capable of increasing and maintaining the state of ketosis compared with ketone salts. Some small clinical trials have compared the two different forms of the same molecule, concluding that the ketone esters were able to increase free BHB levels 50% higher than ketone salts [34]. Recently, supplementation with ketone body esters has shown an improvement in exercise performance [35,36].

A ketone diet is characterized by the supply of approximately 80–90% of calories from fat, 10–15% of calories from protein, and <5% calories from carbohydrates [37], stimulating fat oxidation and promoting fat loss, which are important in obesity conditions [38]. The ketone diet has demonstrated successful results in the treatment of epilepsy and other neurological conditions [39]. However, prolonged ketone diets seem to have an impact on liver steatosis [40,41], glucose homeostasis [40–42], and dyslipidemia [43].

#### **6. Metabolic Functions of Ketone Bodies in Vital Organs**

During low-carbohydrate conditions, ketone bodies are used in proportion to their plasma concentrations, and consequently, liver production.

It has been demonstrated that the acetoacetate generated from an oral intake of BHB esters reaches first the heart, followed by the kidneys, brain, skeletal muscles, and intestine. Normally, heart tissue can oxidize ketones which enter cardiomyocyte cells, thanks to a specific carrier called MCT1 (monocarboxylate transporter) [44]. In the mitochondria of these cardiomyocytes, ketones are converted into AcAc and then into aceto-acetyl CoA, thanks to the activity of the enzyme SCOT. After this conversion, a thiolase transforms the last product into two molecules of acetyl CoA, which enter the TCA cycle for the production of ATP. In stressful situations, such as heart failure and aortic stenosis-induced left ventricular hypertrophy, the circulating levels of ketones and their oxidation states are heightened, supporting the role of ketones in providing the increased energy demanded by the heart [45].

In the ketosis physiological state, BHB can easily enter proximal tubular cells because there is no saturation mechanism [10]. There, BHB is oxidized through the TCA cycle that produces acetyl CoA, which is then transformed via oxidative phosphorylation into ATP.

An adequate and consistent energy supply is necessary to maintain brain cell functions since low glycogen storage is present inside the brain [46]. This is evidenced in pathological conditions with defects in the brain (e.g., glucose transporter type 1 (GLUT-1 deficiency), with impaired cerebral glucose uptake and consequent seizures, movement disorders, and cognitive impairments [47]. The uptake of ketone bodies across the blood–brain barrier (BBB) is possible via monocarboxylate transporter (MCT) [48], with MCT1-isoform expressed by endothelial cells and astroglia [9]. The brain uses ketone bodies, which can give more than 50% of its energy requirements.

Ketone bodies are also important energy substrates for skeletal muscle, with a robust anticatabolic response, reducing phenylalanine efflux from muscle [49].

Ketone bodies derived from short-chain fatty acids are employed by colonic epithelial cells as respiratory fuels where they predominantly use butyrate sequentially followed by acetoacetate, glutamine, and glucose, notwithstanding the interaction of substrates [50]. Ketone body signaling facilitates Lgr5+ intestinal stem cell (ISC) homeostasis aiding in post-injury intestinal regeneration, restoring intestinal regeneration, and resuming ISC function [51].

#### **7. Biological Properties of Ketone Bodies**

The increase in ketone body blood levels derives from fatty acid breakdown during low carbohydrate availability, resulting in a danger signal of starvation and a physiological response for improving survival during starvation. The energy production from ketone bodies is associated with increased radical oxygen species (ROS) release in the mitochondria, an increase in NAD+ levels, and a lower AMP/ATP ratio. Ketone bodies and the ketogenic diet act in upregulating anti-oxidant and anti-inflammatory mechanisms (Figure 3).

**Figure 3.** Molecular pathways involved in ketone bodies effect on oxidative stress, inflammation, and epigenetic control. HDAC: histone deacetylase; BDNF: brain-derived neurotrophic factor; NFkB: nuclear factor kappa-light-chain-enhancer of activated B cells; FOXO3A: forkhead box O3; Mt2: mammalian metallothionein-2; UCP: uncoupling protein; PPARγ: peroxisome proliferator-activated receptor γ; p53: protein 53; Ku70: DNA repair subunit protein; GSK3: serine/threonine protein kinase; mTOR: mammalian target of rapamycin.

Consistently, ketone bodies preserve mitochondria and their role in cellular energy homeostasis [52]. An increased uptake of Ca2+ into mitochondria enhances ROS production and blocks the synthesis of ATP, inducing cytochrome c release and mitochondrial membrane potential [53]. These modifications cause mitochondrial swelling and apoptosis [54], with consequently impaired energy homeostasis [55]. BHB is able to maintain mitochondrial density and function [56] by controlling the mitophagy of damaged mitochondria and inducing the renewal of the mitochondria population [57]. The mitochondrial biogenesis is enhanced via Nrf2 activation, which induces the transcription of PGC-1 (peroxisome proliferator-activated receptor gamma coactivator-1) [58], which controls the transcription of TFAM (mitochondrial transcription factor A), resulting in the replication of mitochondrial DNA and the mitochondrial biogenesis [59].

The block of glutathione peroxidase (GSH-Px), a key rate-limiting enzyme in ROS formation [60] by BHB, reduces the production of ROS, with a consequent improvement in mitochondrial respiration and homeostasis [61–65], ATP production, activation of adenosine receptors that lower oxidative stress [66], upregulation of antioxidant genes and activation of antioxidant enzymes that control lipid peroxidation and protein oxidation [67].

Ketones' anti-inflammatory effects are related to the inhibition of the NLRP3 (NODlike receptor pyrin-domain containing-3) inflammasome, which activates caspase-1 and the release of the pro-inflammatory cytokines (IL-1β and IL-18) [68]. Ketone bodies block the K+ efflux, which activates the NLPR3 inflammasome [69]. During brain injury induced by the middle cerebral artery occlusion (MCAO) model, ketone bodies inhibited NLRP3 and inflammation [70]. In a randomized, controlled dietary intervention trial with 40 overweight subjects aged 18–55 years fed with a diet very low in carbohydrates or an isocaloric diet low in fat for 12 weeks, the subjects following the ketogenic diet showed lower inflammation, with the reduction of interleukin-8 (IL-8), TNF-alpha, plasminogen activator inhibitor-1 (PAI-1), monocyte chemoattractant protein (MCP-1), E-selectin, and intercellular adhesion molecule-1 (ICAM-1) in the presence of mild inflammation [71].

The immune response is controlled by ketones by the binding of BHB to HCAR2 (hydroxycarboxylic acid receptor 2), which results in the induction of prostaglandin D2 (PGD2) production by cyclooxygenase 1 (COX1) [72] and the inhibition of NF-KB (nuclear factor kappa-light-chain-enhancer of activated B cells) mediated inflammation through the blockage of IKB kinase (IKK), by a metabolite of PGD2.

BHB is involved in controlling cellular function via epigenomic regulation. Ketone bodies control histone post-translational modifications, including histone methylation (Kme), histone/lysine acetylation (Kac), and β-hydroxybutyrylation (Kbhb), which regulate chromatin architecture and gene expression. BHB is able to inhibit the histone deacetylase (Class 1 and Class IIa HDACs) leading access to transcription factors of genes encoding oxidative stress resistance factors like FOXO3 (forkhead box O3) and Mt2 (Mammalian metallothionein-2) [73]. The induction via BHB of histone Kbhb levels with site-specific lysine residues (H3K4, H4K8, H3K9, H4K12, H3K56) is increased in human embryonic kidney 293 (HEK293) cells during prolonged fasting, supporting a direct role in chromatin structure and functions regulation [74]. In HEK293 cells transiently transfect with ORM (yeast)-Like protein isoform 3 (ORMDL3) mRNA expression, an asthma susceptibility gene [75], BHB controlled inflammation inhibiting ER stress response pathway proteins and enhancing both Foxp3 and manganese superoxide dismutase (MnSOD) transcription via AMP-activated protein kinase (AMPK) activation, leading to a decrease in cellular oxidative stress [76].

BHB inhibition of HDACs leads to increase expression of brain-derived neurotrophic factor (BDNF), which exerts neuroprotective effects against various insults to the central nervous system, as the functional recovery after traumatic brain injury (TBI) in mice [77].

BHB can act as an inducer of transcription factor Nrf2 (nuclear factor-erythroid factor 2-related factor 2). Nrf2 is a transcription factor that regulates the cellular defense against toxic and oxidative insults through the upregulation of the expression of genes involved in the oxidative stress response and drug detoxification [78]. Human microvascular endothelial cells (HMEC-1) exposed to ketone bodies increased NRF2 expression with a clear translocation to the nucleus and induction of antioxidant proteins [79].

#### **8. Role of Ketone Bodies in Pathophysiological Ailments**

Critical illness has demonstrated various disruptions in metabolism and mitochondrial function. Whether it arises from organ failure or microbial infections, the metabolic response to these ailments requires maintenance of metabolic balance, nutrient utilization for different activities, and functional recovery. On the other hand, a metabolic response can create clinical consequences such as catabolic processes that can impair physiological stability. Furthermore, a long period of metabolic imbalance could produce mitochondrial dysfunction, including energy crisis and high free-radical production, resulting in a compromised immune system, tissue and organ failure, and death [80–83].

Critical-illness-affected patients require an energy source to support physiological stress responses and to give robust protection to critical organs such as the heart, brain, liver, and kidney [81,83] (Figure S1). Additionally, increasing evidence across animal and human studies has shown ketones are a beneficial alternative substrate due to reloading acetyl-coenzyme-A through an independent pathway irrespective of glucose levels [84], and are useful to maintain the cytosolic NAD+ (nicotinamide adenine dinucleotide) pool which is pivotal for cellular survival, antioxidants, and pro-survival pathways [85]. As serum ketone body concentration varies during physiological and pathological conditions and acts as potent endogenous signaling molecules, they may act in cellular protection and repair, mitochondrial biogenesis, antioxidant defenses, and enhanced autophagy [80,82,86–88]. With the known and possible mechanistic properties of ketones, their use as an individual or adjunct therapy for different conditions in critical illness has been explored.
