Ketones in the Life Sciences – Biochemistry, Metabolism, and Medicinal Significances

Abstract

Being very soluble in aqueous solutions with relatively low toxicity and high stability, ketones play central roles in intermediary metabolism and physiological homeostasis. In mammals, lipid catabolism by β-oxidation of fatty acids produces acetyl-CoA, which is converted to ketone bodies in a process known as ketogenesis. During periods of low glucose availability, the synthesis of ketones from lipid sources represents a metabolic shift. Ketone bodies are formed in the hepatic tissues and travel to extrahepatic tissues to serve as an alternative energy source to carbohydrates during periods of fasting, post-exercise, pregnancy, and starvation. This is particularly important to fuel the brain in times of nutritional deprivation. Ketogenesis is hormonally upregulated by glucagon, thyroid hormone, catecholamines, and cortisol. Insulin is the primary negative regulator of this process so that low insulin levels trigger ketogenesis. Ketones can also be involved in other biological processes such as de novo lipogenesis and sterol synthesis, as well as gluconeogenesis, β-oxidation, and tricarboxylic acid cycle. Several inborn errors of metabolism highlight the importance of ketones in energy generation. The ubiquitous nature of ketones, as well as their key roles in regulation of metabolic pathways, makes them attractive targets for new drug development.

1. Introduction

Ketones that have at least one alpha-hydrogen will undergo keto-enol tautomerization (Figure 1), a process catalyzed by both acids and bases. The keto form is usually more stable than the enol. Furthermore, the C−H bonds adjacent to the carbonyl in ketones are more acidic (pKa ≈ 20) than the C−H bonds in similar alkane structures (pKa ≈ 50). This difference reflects resonance stabilization of the enolate ion that is formed upon deprotonation, where the relative acidity of the α-hydrogen is important in the enolization. There are three main ketones which comprise ketone bodies, a distinct group of highly water-soluble compounds which can readily enter the circulation and move into tissues through the interstitial fluids. Of the three ketone bodies, only acetone is a true ketone, but is generally metabolically inert, while acetoacetate is a true ketoacid and β-hydroxybuturate is a hydroxy acid.

Figure 1.

Ketones have at least one alpha-hydrogen that undergoes keto-enol tautomerization with the keto form (left) usually being more stable than the enol form (right).

During periods of caloric restriction, fasting, or extended exercise, hepatic glycogen stores are mobilized to maintain proper glucose levels, but glycogen levels can become depleted within 24 hours of prolonged glucose deprivation. At this point, metabolic switches are triggered to allow the use of alternative energy sources. Insulin levels are low while glucagon and epinephrine levels in the blood are elevated. Under these conditions, ketogenesis is activated and fats stored in adipose tissue are released from the fat cells into the blood as free fatty acids and glycerol. Fatty acids in the bloodstream are taken up by cells and are metabolized by β-oxidation in the mitochondria. The use of fatty acids as high-energy fuels when glucose stores are low is termed “metabolic sparing,” where fatty tissues supply energy to skeletal muscle, myocardium, and liver, sparing the glucose for use in the central nervous system. Release of fatty acids from adipocytes into the circulation can result in an increase in plasma fatty acids and ketone body concentrations by up to 5-fold and 20-fold, respectively. Almost 99% of plasma fatty acids become complexed with albumin and transported by the bloodstream to tissues outside the liver. In contrast, the high aqueous solubility of ketone bodies in interstitial fluids allows them to be readily available to most tissues.

1.1 Ketone bodies

Ketogenesis has three highly regulated control points including lipolysis in adipocytes to generate non-esterified fatty acids or “free fatty acids, entry of these fatty acids into the mitochondria and an irreversible first enzymatic step of ketone body synthesis [1]. The common intermediate in metabolic energy production linking the predominant metabolic pathways is acetyl-CoA (Figure 2), which can be generated by catabolism of lipids, polysaccharides, and proteins. Acetyl-Co generated from lipid catabolism enters the tricarboxylic acid cycle (TCA cycle) allowing ATP generation when glucose is limited. This is the basic premise for ketones as an alternative carbon source for energy production, in response to reduced glucose availability. Under normal well-fed conditions, ketogenesis by the liver is negligible, with plasma ketone body concentrations generally less than 0.5 mmol/l. When nutritional needs are met, glucose and insulin levels are high, while the opposing actions of glucagon are low. Ketone bodies and medium-chain fatty acids (4–12 carbon) may readily pass into the mitochondria to be used for acetyl-CoA production, while long-chain fatty acids depend on a carnityl-transferase system to be ferried across the mitochondrial membrane [2] and is a major control point for lipid metabolism [3, 4, 5].

Figure 2.

Structure of acetyl-coenzyme a with the transferable acetyl group to the far left in blue.

Ketogenesis begins in mitochondrial matrix with acetyl-CoA, which is used as two-carbon building blocks of the 4-carbon ketone bodies produced in the liver (Figure 3). These hepatically-generated ketone compounds include acetone, acetoacetic acid (acetoacetate), and beta-hydroxybutyrate (β-HBT). Acetone is the third ketone body, which is a highly volatile but metabolically inert compound formed as a spontaneous breakdown product of acetoacetate [6].

Figure 3.

The ketogenesis pathway uses mitochondrial acetyl-CoA as substrate for formation of 4-carbon ketone bodies including acetoacetate and β-HBT in a series of reversible reactions. The rate limiting step (*) is catalyzed by HMG-CoA reductase. Non-enzymatic decarboxylation of acetoacetate leads to acetone formation.

Red blood cells do not contain mitochondria and are therefore entirely dependent on anaerobic glycolysis for their energy requirements. In all other tissues, the fatty acids are transported to the mitochondria to enter the β-oxidation pathways forming the energy currency of acetyl-CoA. This is in contrast with the brain, which cannot utilize fatty acids for energy and requires ketone bodies formed from other tissues, as a means of energy transport from lipid stores to the CNS. However, recent evidence suggests that glial cells may help fuel astrocytes with locally synthesized ketone bodies during times of nutrient deprivation [7]. Both acetoacetate and β-HBT can readily cross the blood–brain barrier, as well as the placental barrier to serve as an alternate energy source to glucose. The high solubility in aqueous environments is the single most important physical-chemical aspect of ketone bodies in biological systems. It also allows widespread dispersion to all tissues following synthesis in the liver and rapid metabolic availability due to unencumbered penetration through mitochondrial membrane pores without the use of transport systems.

2. Molecular mechanisms of ketone biochemistry

2.1 Ketogenesis

Ketone body synthesis occurs in the mitochondrial matrix, where the first step of ketogenesis involves the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA in a reaction catalyzed by acetoacetyl-CoA thiolase (thiolase or β-thiolase, EC 2.3.1.9) with regeneration of one reduced HS-CoA molecule (Figure 3). Acetoacetyl-CoA can be further oxidized by HMG-CoA synthetase (HMG-CoA synthetase, EC 2.3.3.10) using a third acetyl-CoA as cofactor, generating β-hydroxy-β-methylglutarate-CoA (or HMG-CoA) in the rate-limiting enzymatic step of ketogenesis. HMG-CoA lyase (EC 4.1.3.4) catalyzes removal of acetyl-CoA to form the first type of ketone body acetoacetate, which can be reduced to β-HBT by β-hydroxybutyrate dehydrogenase (EC 1.1.1.3) with NADH as a cofactor (Figure 3). β-HBT is the most abundant of the ketone bodies. It should be noted that acetoacetate and β-HBT can readily be converted back into acetyl-CoA due to the reversive nature of several ketogenic enzymes in most tissues of the body, with the notable exception of the liver (Figure 3).

2.2 Ketolysis

Circulating acetoacetate produced from ketogenesis (or produced from β-HBT lysis) can interface with the TCA cycle (Figure 4). Acetoacetate can undergo addition of a CoA group by mitochondrial succinate-CoA-3-oxaloacid CoA transferase (SCOT, EC 2.8.3.5) the rate-limiting step in ketolysis through the TCA cycle [8]. SCOT is a homodimer mitochondrial matrix enzyme [9, 10] expressed in all tissues except the liver [11, 12]. The absence of SCOT in the liver prevents the futile cycling of acetoacetate and acetyl-CoA in hepatic tissues. Ketolysis occurs in the non-liver cells, particularly the heart, brain, and skeletal muscles [13] and provides more energy for ATP synthesis than β-oxidation of fatty acids [14]. It is important to reemphasize that fatty acid oxidation and ketogenesis occurs primarily in the liver, whereas ketolysis occurs in non-liver cells and that the absence of the SCOT enzyme in the liver allows ketone body formation to proceed for use in other tissues.

Figure 4.

Acetyl-CoA is the central 2-carbon currency used to feed the TCA cycle. Substrates for acetyl-CoA formation include: Pyruvate derived directly from glycolysis and indirectly from glycogenolysis; acetoacetyl-CoA from ketone bodies by the action of succinyl CoA transferase and then β-thiolase; and from β-oxidation of fatty acids derived from lipolysis. Removal of acetyl-CoA from the cytoplasmic pool can occur through carboxylation by acetyl-CoA carboxylase to form malonyl-CoA committing it to fatty acid synthesis.

Acetoacetate can also spontaneously decarboxylate in an irreversible reaction to form acetone and CO₂ (Figure 3). Acetone can also cross the blood–brain barrier but is metabolically inert. Acetone cannot be directly converted back into acetoacetate or acetyl-CoA. However, the detoxification of acetone occurs in the liver where it is converted into lactic acid, which can, in turn, be oxidized into pyruvic acid, and only then into acetyl-CoA. Acetone is highly volatile and readily expelled from the lungs during respiration with the elimination half-life of acetone is reported to be about 27 hours [15].

3. Acetyl-CoA production

Insulin is an anabolic hormone that promotes the synthesis of protein and glycogen and prevents breakdown of muscle tissue and fat stores. In contrast, glucagon is one of the catabolic hormones which promotes production of glucose-6-phosphate from glycogen breakdown and glucose synthesis through gluconeogenesis (Table 1). This seemingly antagonistic relationship between insulin and glucagon actually assures that glucose, acetyl-CoA, free fatty acids, and ketone levels are adequate to meet energy requirements, at ratios which accurately reflect changing nutritional status and needs. Metabolism of ketone bodies interfaces with the TCA cycle, β-oxidation of fatty acids, de novo lipogenesis, sterol biosynthesis, glucose metabolism, and the mitochondrial electron transport chain. From an energy standpoint, the most important of these interfaces is with TCA cycle. Briefly, the TCA cycle is a central metabolic pathway used to generate energy by organisms that respire in a process termed aerobic metabolism. The TCA cycle (Figure 4) consumes acetate in the form of acetyl-CoA with production of NADH by reduction of NAD⁺. The NADH is fed into the oxidative phosphorylation (electron transport) pathway to form ATP, the predominate energy currency of the cell. However, acetyl-CoA is the pivotal molecule responsible for feeding the TCA cycle being supplied by both the lipolysis and glycolysis pathways which effectively links lipid (Figure 4), protein, and carbohydrate metabolism (Figure 5) as two-carbon intermediates.

Hormone	Targeted Organs	Effect
Glucose metabolism
Insulin	Muscle, Adipose, Liver	Stimulates glucose transport, increases glycogenesis and glycolysis
Glucagon	Liver	Promotes glycogenolysis, gluconeogenesis
Fat metabolism
Insulin	Adipose, Liver	Reduces hormone-dependent lipase activity, activates acetyl-CoA carboxylase, promotes fatty acid synthesis
Glucagon	Adipose, Liver	Activates hormone-dependent lipase, decreases acetyl-CoA carboxylase activity

Table 1.

Effects of insulin and glucagon on metabolic activities in different organs using either glucose or fat as fuel source.

Figure 5.

Acetyl-CoA can be generated during starvation from proteolysis to form ketogenic and glucogenic amino acids. Leucine and lysine are ketogenic and can only be used to form acetyl-CoA from ketogenesis, all others are glucogenic and can be used for acetyl-CoA production or for intermediates of the TCA cycle. Some amino acids can be used for both ketogenic and glucogenic generation of acetyl-CoA.

3.1 Formation of acetyl-CoA by fatty acid β-oxidation

Free fatty acids are negatively charged and cannot directly enter cells without using one of the SCL27 family of fatty acid transport proteins located on the outer cell membrane [16, 17]. The translocation of the fatty acids from the cytoplasm into the mitochondrial matrix depends on a shuttle system with carnitine acyltransferases on both sides of the inner mitochondrial membrane and an acylcarnitine transferase anchored to the membrane. Carnitine acyltransferase enzymes (CAT) catalyze a reaction where the acetyl group of fatty acyl-CoA displaces the hydrogen atom in the central hydroxyl group of carnitine. This reaction is highly reversible and does not depend on the order in which the substrates bind to the enzyme [2]. More specifically, long-chain carnitine acyltransferase (palmitoyl-CoA: l-carnitine O-palmitoyl transferase I (CPT1, EC2.3.1.-) on the outer membrane links medium- or long-chain fatty acyl-CoA molecules to free carnitine to form acylcarnitine, which can diffuse through the porous outer mitochondrial membrane into the intermembrane space (Figure 6). Acylcarnitine can be passively transported across the inner membrane by acylcarnitine translocase in exchange for a free carnitine. When the acyl-carnitine is finally delivered into the mitochondrial matrix, palmitoyl-CoA:l-carnitine O-palmitoyl transferase II (CPT2, EC2.3.1.21) catalyzes the transfer of the fatty acid back to CoA with release of a free carnitine ready to be shuttled back to the mitochondrial intermembrane space [18]. The fatty acyl-CoA is released by CPT2 into the mitochondrial matrix for entry into the β-oxidation pathway. Similarly, short-chain fatty acids (2–6 carbons) are linked to carnitine by corresponding carnitine O-acetyltransferases (EC2.3.1.7) on the outer and inner mitochondrial membranes. Another transferase system, carnitine O-octanoyltransferase (EC 2.3.1.137) can accommodate a wide range of fatty acids. Since most fatty acids are between 16 and 18 carbons, the CPT1/CPT2 is the predominate shuttle for acyl-fatty acids into the mitochondria.

Figure 6.

Cytoplasmic fatty acyl-CoA molecules are shuttled across the mitochondrial membrane using the carnitine acyltransferase (CAT) transport proteins on the membrane surface. Long chain fatty-acyl molecules bind the carnitine palmityl-transferse I (CPT1) on the porous outer membrane removes the CoA and attaches a carnityl group to enter the intermembrane space where carnitine acyl-translocase passively transports the carnityl-acyl molecule across the inner membrane for exchange of a fee carinitine. Carnitine palmityl-transferse II (CPT2) replaces the CoA moiety with release of free carnitine. Once in the mitochondrial matrix, β-oxidation of fatty acylCoA begins.

Insulin stimulation of cells can inhibit β-0xidation of fatty acids by activation of the mitochondrial enzyme acetyl-CoA carboxylase (EC 4.6.1.2). This enzyme catalyzes an irreversible carboxylation of acetoacetyl-CoA to form malonyl-CoA for use as a precursor for fatty acid synthesis (Figure 3). Malony-CoA is a potent inhibitor of CPT1 and inhibits acyl-CoA transportation into the mitochondrial matrix. The CPT1 isoform in the heart/skeletal muscle tissue is 30–100-fold more sensitive than the liver isoform CPTI [19]. In liver, the role of malonyl-CoA as a regulator of CPT1 may depend on the nutritional status. In one study, the Ki of malonyl-CoA on CPT1 was 1.5uM and the sensitivity decreased with continued starvation exhibiting a Ki of 3.0 uM after 18 hours and a Ki of 5.0 after 42 hours [20]. Overall, malonyl-CoA serves as the designated substrate for fatty acid synthesis and inhibits β-0xidation of fatty acids, effectively preventing a futile cycle of acyl-CoA molecule by exhaustive synthesis and oxidation during homeostasis. However, during starvation it appears that malonyl-CoA may become less effective at inhibiting CPT2 activity, allowing more β-oxidation of fatty acids to occur.

Once inside the mitochondrial matrix, β-oxidation of acyl-CoA is carried out in a four-step process (Figure 7). Briefly, acyl-CoA is dehydrogenated by acyl-CoA dehydrogenase (EC 1.3.99.3) to introduce a C2–3 double bond with FAD⁺ as the electron acceptor forming FADH₂ and a molecule of trans-delta-2 enoyl CoA. The double bond is hydrated by enoyl-Co hydratase (EC 4.2.1.17) to form l-3-hydroxyacyl CoA. The hydroxyl group on the C3 is dehydrogenated by 3-hydroxyacyl CoA dehydrogenase (EC 1.1.1.35) using NAD⁺ as the electron acceptor to form NADH and 3-keto acyl-CoA. The final reaction in the β-oxidation cycle is catalyzed by β-thiolase enzyme (also called acetyl-CoA acetyltransferase) which cleaves the C2–3 double bond releasing newly formed acetyl-CoA and an acyl-CoA ready for the next cycle (Figure 7). It should be noted that the last three steps are catalyzed by a hormonally responsive ‘mitochondrial trifunctional protein,’ which includes the hydratase, dehydrogenase, and thiolase enzyme activities [21, 22]. The β-oxidation cycle is repeated until all carbons of the fatty acyl-CoA are converted into acetyl-CoA.

Figure 7.

β-Oxidation of fatty acyl-CoA molecules proceeds in a four-step enzymatic process which can be repeated in cycles to remove 2-carbon units to form acetyl-CoA until the hydrocarbon chain is degraded.

3.2 Formation of acetyl-CoA through glycolysis

Glycolysis is the anaerobic breakdown of carbohydrates (monosaccharides), ending with the pyruvate decarboxylation catalyzed by pyruvate dehydrogenase complex (EC 1.2.4.1) to form acetyl-CoA and CO₂ in a CoA- and NAD-dependent reaction. Glycogenolysis provides glucose-6-phosphate to feed into the glycolysis pathway. Pyruvate dehydrogenase catalyzes a crucial step in metabolism, as it allows acetyl-CoA formed from the anaerobic glycolysis pathway to enter the TCA cycle (Figure 4) allowing cell respiration [23]. Condensation of acetyl-CoA with oxaloacetate catalyzed by citrate lyase forms citrate which is used directly in the TCA cycle. The shuttling of two carbon units through acetyl-CoA provides a basic framework for energy production from breakdown of both fats and sugars in the body, where the lack of carbohydrate can be compensated by use of fats as a main energy source. Other nutrients can be used as gluconeogenic precursors including glycerol, lactate, pyruvate, certain amino acids, and odd-chain length fatty acids [24].

3.3 Formation of acetyl-CoA with amino acids

Although lipids are the primary source of acetyl-CoA, skeletal muscle proteins can be broken down into peptides then into amino acids to be used for both glucose and ketone body formation (Figure 5). The various metabolic pathways for conversion of each amino acid are beyond the scope of this review. Briefly, glucogenic amino acids (including all amino acids except leucine and lysine) are used to form glucose in a process that involves hepatic conversion to alpha ketoacids and then to glucose. This is the predominate catabolic pathway and accelerates during periods of increased fasting and starvation. Conversely, ketogenic amino acids (including isoleucine, leucine, lysine, phenylalanine, tryptophan, and tyrosine) are converted to ketone bodies which can be used in extrahepatic tissues [25, 26].

If carnitine concentrations are high, acetyl-CoA generated from pyruvate by pyruvate dehydrogenase is readily conjugated to carnitine by carnitine-acetyltransferase for exportation to other tissues [27]. Alpha-keto acids formed during mitochondrial catabolism of lysine and the branched amino acids in the liver and kidney (valine, leucine, and isoleucine) can also be conjugated to carnitine. These and other conjugated organic acids are exported as short-chained acylcarnitine molecules into circulation [28, 29, 30]. These conjugation reactions are important for short-chained fatty acids formed from partial β-oxidation, whose trans conjugation from-CoA to carnitine will free up the-CoA coenzyme for further use in the TCA cycle or additional cycles of conjugation. This may be particularly important in the cardiac and skeletal muscle, where short-chain fatty acid oxidation is less efficient than for long-chain fatty acids. As discussed earlier, free carnitine is also needed for transport of fatty acids into hepatic mitochondrial matrix [31]. As a side note, large doses of carnitine (2–5 g/day) are used by many athletes for performance enhancement [32, 33], and lower the blood lipid levels in hyperlipidemic or diabetic patients [34, 35, 36].

4. Rate-limiting steps in ketone synthesis and utilization

4.1 Regulation of key enzymes

The production of ketone bodies by conversion of acetyl-CoA to acetoacetate and β-HBT is catalyzed by a series of reversible enzymatic steps (Figure 4). HMG-CoA synthetase and HMG-CoA lyase activities represent the rate-limiting enzymatic steps in ketone body formation in the hepatic mitochondria. However, there are several other enzymes that are key regulatory steps in ketone body formation and utilization, most of which are involved in mechanisms used to switch from anabolic to catabolic metabolism. As stated earlier, global regulation of intermediary metabolism occurs in the body through the opposing actions of anabolic hormones (mainly insulin) and catabolic hormones (mainly glucagon, cortisol, and adrenaline) (Table 1). Cortisol and adrenaline are known as stress hormones, which promote survival of the organism by mobilizing energy sources and downregulating metabolic processes which are not immediately necessary [37]. Glucagon triggers the most important change in the systemic metabolism during fasting, which involves the mobilization of lipids stored in adipose tissue and break down of triglycerides to free fatty acids and glycerol [38, 39]. On the other hand, insulin rapidly inhibits lipolysis in skeletal muscle at low physiological levels [38]. The key points in hormonal regulation of ketone synthesis involve several enzymes including hormone-sensitive lipase (HSL), acetyl-CoA carboxylase, succinyl CoA-oxoacid transferase (SCOT), and HMG-CoA synthase. The importance of these control points is emphasized by a number of inborn errors of metabolism, where enzyme defects manifest as diseases of fatty acid and ketone metabolism.

Hormone-sensitive lipase (HSL, EC 3.1.1.79) is a cytosolic enzyme found in adipose tissue and catalyzes release of free fatty acids from triglyceride stores following hormonal activation. Catabolic hormones including glucagon, epinephrine, Thyroid-Stimulating Hormone, and ACTH bind to G-protein-associated cell surface receptors leading to protein kinase A activation and phosphorylation of HSL. Activated HSL catalyzes lipid molecule breakdown by hydrolysis of a fatty acid from either a diacylglycerol or triacylglycerol molecule releasing a free fatty acid and/or monoglyceride, with an 11-fold higher affinity for the former [39, 40]. HSL allows mobilization of lipid-derived energy stores and plays a major role in ketogenesis in most cells [41, 42]. This enzyme is a key “on switch” to ketogenesis which is regulated by glucagon (+) and insulin (−) signals.

Acetyl-CoA carboxylase (EC 6.4.1.2) is a biotin-dependent cytoplasmic enzyme whose main function is to provide malonyl-CoA precursors for fatty acid synthesis (Figure 5). When activated by insulin, acetyl-CoA carboxylase catalyzes irreversible carboxylation of cytosolic acetyl-CoA to form malonyl-CoA (Figure 3) using one molecule of bicarbonate and ATP [43]. Malonyl-CoA provides 2-carbon fatty acid units, committed to fatty acid chain elongation during lipid biosynthesis. As discussed earlier, malonyl-CoA can also inhibit the transfer of the fatty acyl group from acyl-CoA to carnitine catalyzed by carnitine acyltransferase in the cytoplasm. This inhibits the transportation of fatty acids into the mitochondrial matrix, effectively blocking β-oxidation fatty acid breakdown and reducing acetyl-CoA needed for ketogenesis. Acetyl-CoA carboxylase is found in the cytoplasm of all cells and is enriched in tissues where fatty acid synthesis is important such as adipose tissue and mammary glands [44]. This enzyme is a key “off switch” to ketogenesis which is regulated by insulin (+) and glucagon (−) signals. As mentioned earlier, malonyl-CoA inhibition of CPT1 transferase in mitochondrial prevents futile cycling of acetyl-CoA derived from lipolysis.

Succinyl CoA synthetase EC 6.2.1.4 GTP forming; EC 6.2.1.5 ATP forming) is an enzyme of the mitochondrial matrix that plays a dominant role as one of key steps in the TCA cycle. It catalyzes the reversible transfer of coenzyme A from succinyl-CoA to succinate, with formation of a nucleotide triphosphate (GTP or ATP) by substrate-level phosphorylation: Succinyl CoA + Pi + NDP ↔ Succinate + CoA + NTP [45, 46]. This is similar to the activity of SCOT, where acetoacetate receives the-CoA from succinyl CoA with production of acetoacetyl-Co and succinate [47], so that acetoacetyl-CoA can be metabolized to acetyl-CoA and succinate can progress through the TCA cycle for conversion to oxaloacetate (Figure 4). As a feedback control, increased levels of acetoacetate in the mitochondria of target organs inhibit succinyl CoA synthetase and therefore inhibit ketone metabolism [1]. Succinyl CoA synthetase also has a role in porphyrin and heme production by controlling the levels of their precursor succinyl CoA [48]. Control of these enzymes has the effect of slowing down ketone production in the presence of insulin and increasing it in the presence of glucagon.

Hydroxymethylglutaryl-CoA synthase (HMG-CoA synthase, EC 2.3.3.10) is a mitochondrial enzyme, which catalyzes a reversible reaction that condenses acetyl-CoA with acetoacetyl-CoA to form HMG-CoA (Figure 3). HMG-CoA is an intermediate in both cholesterol synthesis and as the second step in the mevalonate-dependent isoprenoid biosynthesis pathway. Activation of HMG-CoA synthetase causes shunting of excess acetyl-CoA into the ketone synthesis pathway via HMG-CoA [49]. As stated earlier, this enzyme is inhibited by insulin and becomes over-activated in uncontrolled type I diabetes when insulin levels are low. This causes shunting of acetyl-CoA into ketone synthesis and an exhaustion of substrates for gluconeogenesis (notably oxaloacetate) and excessive HMG-CoA synthetase activation is a key determinant in the development of diabetic ketoacidosis.

4.2 Inborn errors of ketone metabolism

Succinyl-CoA synthetase (SCS) exists in two forms in mammals, defined by the specific nucleotide triphosphate that is generated, with EC 6.2.1.5 as ATP forming and EC 6.2.14 at GTP forming [50]. The enzyme is a heterodimer of α- and β-subunits encoded by two genes, the SUCLG1 encodes the common α-subunit, while β-subunits are coded by SUCLG2, which is GTP-specific and SUCLA2, which is ATP-specific. Β-subunit variants are produced at different amounts in various tissues causing dissimilar GTP or ATP substrate requirements [50]. The heart and brain are energy-consuming tissues and have more ATP-specific succinyl-CoA synthetase, while synthetic tissues such as kidney and liver have the more GTP-specific form [51, 52]. Fatal infantile lactic acidosis is due to a defective SCS ligase function in the SUCL1 gene where patients display a two base pair deletion affecting the α-subunit. This causes a disease in infants that is characterized by the build-up of toxic levels of lactic acid, severe cases can result in death usually within 2–4 days after birth. Without a functional SCS enzyme, there is no operational TCA cycle so that cells must use glycolysis with lactic acid production as the primary means of producing ATP [53].

Succinyl CoA:3-ketoacid CoA transferase (SCOT) deficiency is a rare inherited metabolic disease caused by mutation of the OXCT1 gene which codes for a mitochondrial enzyme SCOT EC 6.2.1.4 responsible for transferring the CoA moiety from succinyl CoA to acetoacetate to form acetoacetyl-CoA and succinate (Figure 4). This impairment of the body’s ability to break down ketones through the TCA cycle leads to an accumulation of acetoacetate. Sequalae include ketoacidotic attacks occurring in one-half of affected individuals during the first 4 days of life and are characterized by extreme tiredness (lethargy), appetite loss, vomiting, rapid breathing, and, occasionally, seizures and coma. Patients usually have a permanently elevated level of ketones in their blood (persistent ketosis) with no symptoms of the disorder between ketoacidotic attacks. This condition is exacerbated by infections, fevers, or periods without food (fasting) and the frequency of ketoacidotic attacks varies among affected individuals [12, 54, 55, 56, 57].

Beta-ketothiolase deficiency is a rare, autosomal recessive metabolic disorder in which the body cannot properly process the amino acid isoleucine or the products of lipid breakdown. It is inherited in an autosomal recessive pattern and is extremely rare with only 50 to 60 cases reported worldwide. The typical age of onset for this disorder is between 6 months and 24 months, where signs and symptoms include vomiting, dehydration, trouble breathing, extreme tiredness, and occasionally convulsions. The inability to metabolize ketone bodies can cause ketoacidotic attacks and even lead to coma. Specifically, there is an accumulation of α-methyl-α,β-keto-butyrate, and α-methyl-β-OH-butyrate (upstream metabolites of the affected enzyme) which may be detected on urine organic acid analysis by GC–MS. Mutations in the ACAT1 gene which encodes the mitochondrial acetoacetyl-CoA thiolase may reduce or eliminate its activity [58].

Medium-chain acyl-CoA dehydrogenase deficiency (MCAD), while not really classified as inborn errors of ketone metabolism, represents an inborn error of lipid metabolism that can manifest as diseases of ketone metabolism. These involve an inability to effectively metabolize medium-chain and long-chain fatty acids. Medium-chain acyl-coenzyme A dehydrogenase EC1.3.8.7 is a mitochondrial enzyme that catalyzes the first step in β-oxidation reactions which converts medium-chain (4–12 carbon) fatty acyl-CoA molecules into acetyl-CoA in the mitochondria for utilization. Acyl-CoA dehydrogenation leads to the formation of the corresponding α,β-unsaturated derivatives using a flavoprotein as follows: a medium-chain acyl-CoA + FAD+ ↔ a medium-chain trans-2,3-dehydroacyl-CoA + FADH [59]. MCAD has an autosomal recessive inheritance pattern, where both parents are asymptomatic carriers. Specific mutations are most commonly of the ACADM gene on chromosome 1. MCAD occurs in about every 1 in 10,000 white infants, with newborn screening done routinely in the United States and many other countries. These patients commonly experience weakness and loss of energy, low blood glucose, and vomiting, which may progress to seizures, comas, hypoketotic hypoglycemia, and failure to thrive if not diagnosed and addressed early. The mainstay of treatment for these patients is early diagnosis and management of diet and lifestyle including the avoidance of fasting [60, 61].

Systemic primary carnitine deficiency (CDSP) is another inborn error of lipid metabolism, which manifests as a defective fatty acid transport system, resulting from an autosomal recessive disorder of carnitine transportation across the plasma membrane specifically the OCTN2 organic cation transporter type 2. The gene responsible for the OCTN2 carnitine transporter is SLC22A5 and is regulated by peroxisome proliferator-activated receptor alpha. The OCTN2 transporter is located in the apical membrane of the renal tubular cells, where it is involved in tubular reabsorption and recapture carnitine prior to its excretion in urine, leading to massively increased urine carnitine levels and significantly reduced plasma carnitine levels in patients with CDSP β. As discussed previously, carnitine is an amino acid necessary for transportation of fatty acids in the mitochondria and when carnitine cannot be transported into the tissues, fatty acid oxidation becomes impaired [62].

Symptoms of CDSP include chronic muscle weakness, cardiomyopathy, hypoglycemia, and liver dysfunction. Diagnosis is established by demonstration of low plasma free carnitine concentration (<5 μM, normal 25–50 μM), reduced fibroblast carnitine transport (<10% of controls), and molecular testing of the SLC22A5 gene [61]. The frequency of CDSP in the United States is estimated to be approximately 1 in 50,000 individuals based on newborn screening data but may vary by ethnicity. Patients can present as infants with hyperammonemia, hypoglycemia, and hepatomegaly or they may present as adults with elevated liver enzymes and hypoketotic, hypoglycemic episodes. CDSP disease may be diagnosed by genetic testing or by checking the carnitine level of the blood. The clinical manifestations include episodes of hypoketotic hypoglycemia, hepatomegaly, elevated transaminases, and hyperammonemia in infants. Typical sequelae from childhood to adulthood include skeletal myopathy, elevated creatine kinase, and cardiomyopathy, arrhythmias, or fatigability. Treatment of these patients includes supplementing carnitine (oral levocarnitine (L-carnitine) at a dose of 50–400 mg/kg/day divided into three doses), reducing fats in the diet, and decreasing fasting durations. Long-term prognosis is favorable if patients remain on carnitine supplementation [61].

5. Medical aspects of ketosis

5.1 Ketoacidosis

Ketogenesis can occur during various scenarios of caloric restriction, including low food intake (fasting), carbohydrate-restrictive diets, starvation, prolonged intense exercise, alcoholism, or during inadequately treated type 1 diabetes mellitus. Under normal well-fed conditions, the concentration of ketone bodies is very low (<0.3 mmol/l) as compared with glucose (∼4 mmol/l). During prolonged fasting, the ketone bodies in plasma may be 2–3 mmol/l, leading to the preferential oxidation and sparing of glucose for use by the brain. This occurs because glucose and ketone bodies have a similar kM for transport to the brain. Ketone bodies begin to be utilized as an energy source by the CNS when they reach a concentration of about 4 mmol/l, which is close to the Km for the glucose by the monocarboxylate transporter [1, 63]. Ketogenic diets can cause what is termed as “physiological ketosis” with ketone bodies increasing to 7-8 mmol/l, whereas uncontrolled diabetic ketoacidosis can cause ketone bodies to reach 20 mmol/l [64, 65, 66]. Due to favorable transport kinetics, ketone bodies are a preferred fuel in the brain during starvation, as acetoacetate and β-HBT readily cross the blood–brain barrier and are used when glucose levels are inadequate. After prolonged starvation, ketone bodies may provide up to two-thirds of the brain’s energy requirements.

Ketoacidosis is a metabolic state associated with pathologically high serum and urine concentrations of ketone bodies, with diabetic ketoacidosis typically following periods of hyperglycemia with relative or absolute insulin deficiency [67]. Diabetic ketoacidosis occurs more frequently in patients with type 1 diabetes, with 10 to 30% of cases occurring with patients with type 2 diabetes. Low insulin levels may occur with diabetic patients, low insulin levels are secondary to hypoglycemia in alcoholic or starvation conditions which result in ketoacidosis. Low insulin levels allow unopposed lipolysis and free fatty acid oxidation resulting in ketone body production and subsequent increased anion gap metabolic acidosis. More specifically, an unfavorable ratio of insulin to glucagon activates hormone-sensitive lipase, which breaks down triglycerides in peripheral fat stores, with subsequent generation of large quantities of acetyl-CoA in more severe forms of both diabetic and alcoholic ketoacidosis. With saturation of the oxidative capacity of the Krebs cycle, a spillover entry of acetyl-CoA into the ketogenic pathway and subsequent generation of ketone bodies occurs. Because ketone bodies are present as unmeasured anions, an increased anion gap metabolic acidosis can occur [68].

While nutritional ketosis and ketoacidosis both involve elevated ketone body levels, diabetic ketoacidosis occurs due to insulin deficiency, with unregulated increases of counterregulatory hormones such as glucagon. This results in unopposed gluconeogenesis by the liver, which elevates blood glucose levels. The surrounding tissues, however, are unable to utilize any of the surplus glucose due to a lack of insulin. In addition to lipid breakdown, amino acid catabolism is also used to meet the energy requirements of the peripheral tissues. When the accumulation of ketone bodies significantly outpaces renal excretion, metabolic acidosis can ensue [68].

Ketoacidosis can also occur in individuals who undergo fasting for extended periods of time [69]. The postabsorptive phase occurs during the first 24 hours of fasting, where dietary deficiency of carbohydrates leads to reduced insulin and elevated glucagon activity. Glucagon stimulates the release of glucose from glycogen stores located in the liver, with hepatic glycogenolysis providing approximately 75% of the required glucose in the postabsorptive phase [70]. The subsequent generation of acetyl-CoA through lipid oxidation exerts an inhibitory effect on pyruvate dehydrogenase, ensuring that pyruvate being formed through glycolysis is utilized as a substrate for gluconeogenesis [71]. Once the glycogen stores are fully depleted, the gluconeogenic phase begins with production of large quantities of gluconeogenic precursors derived from amino acids which are added to lactate, pyruvate, and glycerol to meet cerebral glucose requirements. Further decreases in insulin levels facilitate proteolysis in the muscle tissue, which provides the necessary amino acid substrate supply for increasing hepatic gluconeogenesis, while fatty acid degradation continues [69]. Increased gluconeogenesis leads to depletion of oxaloacetate, which is essential for acetyl-CoA to enter the TCA cycle. As mentioned before, this accumulation of acetyl-CoA inhibits pyruvate dehydrogenase, enhancing pyruvate use in the TCA cycle.

The enhanced production of ketones allows for a protein conservation phase, to spare muscle tissue but allows for energy production [72]. Glucose use in the brain falls from 120 g per day in the first 24 hours of food deprivation to approximately 40 g per day following several weeks of starvation [64]. The breakdown of skeletal muscle tissue decreases from 75 to about 20 g per day [73], as ketone bodies inhibit proteolysis in muscle cells as an adaptive mechanism for prolonged fasting [74, 75]. Gluconeogenesis in the liver proceeds using the lactate, pyruvate, and glycerol produced during continued lipolysis. The kidney medulla, red blood cells, and bone marrow rely solely on this glucose with energy production through glycolysis to generate pyruvate and lactate. These are recycled in the liver to glucose through the Cori cycle, with approximately 40 g of glucose recycled daily with no protein catalysis [69].

Allowing for small losses of ketones in the urine, equal use of ketone bodies in the brain, muscle, and kidney will occur during the protein conservation phase. During starvation ketosis, a steady state plasma bicarbonate concentration of approximately 18 mEq/l, a β-HBT concentration of 8–10 mmol/l will develop, with a normal-to-low plasma glucose concentration. This equilibrium will be maintained by stimulation of insulin release by ketone bodies, as well as their inhibitory effects on lipolysis. This will prevent excessive mobilization of fatty acids from adipocytes [75, 76, 77]. Continuation of the protein conservation phase sees a progressive substitute of keto acids and fatty acids as the preferred energy source for both skeletal and cardiac muscle, where eventually fatty acids become dominant to spar the keto acids for the brain. It is thought that this preferential use has more to do with brain/carcass ratio than actual preferential use of ketoacids rather than fatty acids in the carcass [78].

5.2 Ketogenic diets

Ketogenic diets have enjoyed popularity in both therapeutic and weight control roles [66, 79, 80, 81, 82, 83]. Briefly, a ketogenic diet is an eating pattern consisting of high amounts of fat, adequate amounts of protein, and very low amounts of carbohydrates to less than 50 g/day which in turn leads to preferential formation of ketone bodies or “nutritional ketosis.” However, the level of ketone body concentrations is on the order of 0.5–5 mM compared to 15–25 mM found in “pathological ketosis” [66]. Due to low carbohydrate intake, glycolysis decreases so that oxaloacetate availability for the TCA cycle decreases dramatically. Ketogenesis ensues so that ketone bodies can be utilized as an energy source for the brain, heart, and muscle tissue in the relative absence of glucose, as stated previously. Nutritional components of the classic ketogenic diets are calculated as a ratio of grams of fat to grams of carbohydrate plus protein. The most common ratio is either 3:1 or 4:1, meaning that 90% of the caloric energy comes from fat and 10% from carbohydrate and protein combined [84]. Variations of the classic ketogenic diet have been developed and popularized over the past several decades, including the medium-chain triglycerides (MCT) ketogenic diet, the modified Atkins diet, and the low glycemic index treatment. These alternatives offer more dietary and nutritional flexibility and reduce common side effects associated with ketosis [84, 85, 86].

Ketogenic diets have been used for treatment of refractory epilepsy in children for decades [87, 88, 89]. There may even be a beneficial effect for ketogenic diets in some adults with epilepsy, with less strict diets such as the Atkin’s diet having nearly equal effectiveness [90, 91]. While most dietary fat is composed of long-chain fatty acids, medium-chain fatty acids are more ketogenic. For example, coconut oil is rich in medium-chain fatty acids with around half the calories. With lower dietary calories as fat, a higher portion of carbohydrate or protein can be consumed allowing more food choices [92, 93].

Recent work suggests that metabolic switching that occurs during fasting or extended exercise may be accompanied by adaptive changes in neural networks, which could enhance functionality and resistance to stress, injury, and disease. In this scenario, repetitive cycles of metabolic switching followed by recovery periods may optimize brain function and resilience during the lifespan, with a focus on cognition and mood [94]. In addition, ketone bodies may have a role in regulating several anti-inflammation cellular pathways in patients with dementia and diabetes and improving glucose metabolism, insulin action, and synaptic plasticity, thereby being neuroprotective [95, 96]. Possible therapeutic uses for the ketogenic diet have been studied for many additional neurological disorders [97, 98], including Alzheimer's disease [95], Parkinson's disease [99, 100], traumatic brain injury [101], amyotrophic lateral sclerosis [102, 103], headache, neurotrauma, pain, and sleep disorders [97].

Side effects of ketogenic diets may include constipation, elevation of serum cholesterol, growth slowing, acidosis, and kidney stones [104]. While severe ketoacidosis and electrolyte imbalance can occur in some cases, the risk is virtually nonexistent in persons with normal insulin function, as the concentration of ketone bodies rarely rise above 8 mmol/L. It is important to screen patient for disorders of fatty acid oxidation, especially in children with seizure disorders and developmental abnormalities. Because of the role of acetyl-CoA in isoprene synthesis and heme production, ketogenic diets are contraindicated in patients who disorders in heme biosynthesis such as porphobilinogen deaminase deficiency. Ketogenic diets are also contraindicated in patients with deficiency of pyruvate carboxylase enzyme [105] as this reduces formation of oxaloacetate which is necessary for conversion of acetoacetyl to acetyl-CoA by thiolase.

Measurement of the ketone body levels of beta-hydroxybutyrate and acetone generated during ketosis can provide key information to optimize disease treatment and weight management [67, 106]. Ketone body levels are about 0.1 mM for beta-hydroxybutyrate with about 1 part per million for acetone in the breath of healthy individuals. Their excretion in urine is very low and undetectable by routine urine tests (Rothera’s test) [107]. Dramatic increases of these ketones occur as a consequence of a disease process or when deliberately elevated for treatment of disease [106]. For example, heart failure and ketoacidosis can affect caloric intake and macronutrient management, causing elevation of beta-hydroxybutyrate 30-fold and breath acetone 1000-fold [106]. Measurement of beta-hydroxybutyrate in the blood is the current standard for assessing ketosis. However, the measurement of acetone in the breath can be used for an accurate reflection of the body’s ketone level, as it is both non-invasive and convenient [107]. Acetone is formed by enzymatic and spontaneous decarboxylation of acetoacetic acid and easily crosses the membrane barrior of the alveoli of the lung in the airway for expiration. The most accurate detection of ketone levels in breath is based on mass spectrometer methods, especially selected ion flow tube-mass spectrometry, which is currently the most proven technology [108].

6. Biosynthetic pathways depending on ketones

6.1 Isoprenoids (terpenoids)

The mevalonate pathway uses cytoplasmic acetyl-CoA for biosynthesis of the two 5-carbon building blocks isopentyl pyrophosphate and dimethylallyl pyrophosphate [109]. These two compounds are used to make naturally occurring terpenes (often called isoprenoids), a diverse class of naturally occurring 5-carbon compounds based on isoprene. Isoprene subunits are found in plant derivatives are sometimes used interchangeably with “terpenoids,” but terpenoids contain additional functional groups, usually containing oxygen. Terpenoids comprise about 80,000 compounds, of which 30,000 are biomolecules such as cholesterol, retinol, vitamin K, carotenoids, quinones, coenzyme Q10, and lanosterol derivatives (e.g., steroid hormones), the prenyl chains of chlorophyll, and polyisoprene units of natural rubber [110, 111, 112]. Terpenoids are the largest class of plant secondary metabolites, representing about 60% of known natural products [113], many having substantial pharmacological activity [114] including the anticancer drug paclitaxel. Many plant terpenoids have aromatic or flavoring qualities, including eucalyptus, cinnamon, cloves, and ginger. Several well-known terpenoids have been used in medicinal and traditional herbal remedies including citral, menthol, camphor, salvinorin A in the plant Salvia divinorum, ginkgolide and bilobalide found in Ginkgo biloba, and the cannabinoids found in cannabis [115, 116]. Malonyl-CoA is required for synthesis of flavonoids and related polyketides, for elongation of fatty acids to produce waxes, cuticle, and seed oils in members of the Brassica family, and for malonation of proteins and other phytochemicals [117].

6.2 Cholesterol and steroid synthesis

The rate-limiting step of cholesterol synthesis is the formation 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) where acetyl-CoA condenses with acetoacetyl-CoA in a reaction catalyzed by cytosolic HMG-CoA synthetase [118, 119]. HMG-CoA is enzymatically reduced to mevalonate using NDAPH, which is enzymatically phosphorylated to mevulonate-5-phosphate, an essential intermediate of the isoprenoids. Since HMG-CoA reductase is the rate-limiting step of a complex 37-step process in cholesterol biosynthesis, it became a primary target for pharmaceutical intervention, leading to the development of the statin class of drugs [120]. Cholesterol can act as a structural component of cellular membranes, or it can be used to synthesize steroid hormones, bile salts, and vitamin D [121].

7. Conclusion

Ketones are ubiquitous in nature and are an important part of biosynthetic pathways for a variety of low molecular weight metabolic intermediates as well as complex biological molecules. Their high solubility, easy formation, and relatively low toxicity make ketones very valuable as chemical and biological intermediates in synthetic chemistry. Formation of ketone bodies using acetyl-CoA allows the interconnection of most pathways of energy-generating metabolism including the TCA cycle, β-oxidation of fatty acids, de novo lipogenesis, sterol biosynthesis, glucose metabolism, and the mitochondrial electron transport chain. The role of ketogenesis in diets is a key frontier for therapy of many disease states including obesity, diabetes, inflammation, and neurological disorders. Ketoacidosis in inadequately treated type I diabetes patients remains an area of concern in current medical practice. Because of the vast variety of terpenoids found in nature, it is plausible that many new therapeutic agents will be discovered from these various compounds.

Conflict of interest

The authors declare no conflicts of interest.