International Journal of Molecular Sciences

Review The Chemistry of the : Updates and Opportunities in Organic Synthesis

Michael Scott Williams * and Edward Turos *

Department of Chemistry, University of South Florida, Tampa, FL 33620, USA * Correspondence: [email protected] (M.S.W.); [email protected] (E.T.)

Abstract: The high-fat, low- (ketogenic) diet has grown in popularity in the last decade as a weight loss tool. Research into the diet’s effects on the body have revealed a variety of other health benefits. The use of exogenous supplements to confer the benefits of the diet without strict adherence to it represents an exciting new area of focus. Synthetic ketogenic compounds are of particular interest that has received very little emphasis and is an untapped area of focus for chemical synthesis. In this review, we summarize the chemical basis for ketogenicity and opportunities for further advancement of the field.

Keywords: ketogenic diet; ; keto; ; supplements; acetoacetate; β-hydroxybutyrate

1. Introduction   The ketogenic diet, a high-fat, low-carbohydrate diet, has a long history of use begin- ning primarily as a treatment option for epilepsy [1,2]. However, it is only in the last few Citation: Williams, M.S.; Turos, E. decades that the diet has been popularized among the general public. Renewed clinical The Chemistry of the Ketogenic Diet: interest in the diet and its emerging popularity as a weight-loss tool have led to a larger Updates and Opportunities in scope of research into the diet’s effects on the body and the discovery of a broad range of Organic Synthesis. Int. J. Mol. Sci. physical, biochemical, and cosmetic benefits. 2021, 22, 5230. https://doi.org/ Even with expanding research interest in the ketogenic diet, most prior reported 10.3390/ijms22105230 studies have been done in clinical settings or in biology labs [3,4]. Emerging research shows that certain ketogenic supplements can confer some of the benefits of the ketogenic Academic Editor: Roberto Bei diet while following a normal diet [5,6]. Difficulty in adherence to the ketogenic diet is often cited as a reason why it is abandoned due to limited food options. If these obstacles Received: 29 April 2021 can be overcome, a wide array of possibilities may open up. Yet, very little work has Accepted: 12 May 2021 Published: 15 May 2021 been done so far in expanding the options to do this, particularly from the chemistry side. This review will cover the history and health-related aspects of the ketogenic diet and its biochemical basis as well as highlight some exciting opportunities in organic synthesis to Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in devise routes to potential new ketogenic compounds as components or supplements of the published maps and institutional affil- ketogenic diet. iations. 2. History of the Ketogenic Diet Dietary has been a common societal practice in various religions for thousands of years, and as a treatment for epilepsy for over a century. Much of the early reported information is based on the personal experience of cultists and physicians instead of Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. scientifically motivated clinical trials. In 1911, two French physicians Guelpa and Marie This article is an open access article published a report detailing their use of fasting to treat epileptic seizures, in which six of distributed under the terms and their 26 patients showed a reduction in the severity of their seizures or the rate of seizure conditions of the Creative Commons occurrence [7]. No further details were provided. At the American Medical Association Attribution (CC BY) license (https:// convention in 1921, Geyelin presented a report of a controlled study involving 36 epileptic creativecommons.org/licenses/by/ patients who used fasting as a treatment plan [8]. The results showed that 80% of these 4.0/). patients experienced a decrease in the number of seizures. Despite this promising outcome,

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patients who used fasting as a treatment plan [8]. The results showed that 80% of these patients experienced a decrease in the number of seizures. Despite this promising out- come, fasting saw sporadic use among physicians to try to control epileptic seizures, but Int. J. Mol. Sci. 2021, 22, 5230 no formal clinical trials were done and its use as a long-term trea2tment of 18 method was obvi- ously not viable. Around the same time, research into the use of diet modification to control fasting saw sporadic use among physicians to try to control epileptic seizures, but no formalmellitus clinical was trials developing. were done and The its use difference as a long-term between treatment type method 1 and was obviouslytype 2 diabetes was not un- notderstood viable. at that time and so there was no distinction made. Newburgh and Marsh re- portedAround the the use same of time,a high-fat, research low-carbohydrate into the use of diet modification diet to tomanage control diabetes diabetics in 1920 [9–11]. In mellitus was developing. The difference between type 1 and was not understood1921, Woodyatt at that time published and so there a review was no distinctionarticle on made. the modification Newburgh and Marshof diet to control diabetes reportedmellitus the based use of a on high-fat, his own low-carbohydrate research dietand to that manage of diabeticsPhilip inShaffer, 1920 [9– 11who]. In sought to study the 1921,relationship Woodyatt publishedbetween a acetoacetic review article onacid the and modification glucos ofe in diet vitro to control [12,13]. diabetes It had long been known mellitus based on his own research and that of Philip Shaffer, who sought to study the β relationshipthat diabetic between patients acetoacetic often acid had and glucosean increain vitrosed[12 concentration,13]. It had long been of knownacetoacetic acid, (R)- -hy- thatdroxybutyric diabetic patients acid, often and had an increased in their concentration blood (ketonemia) of acetoacetic and acid, urine (R)-β- (ketourea) based on hydroxybutyricresearch done acid, by and Gerhardt acetone in in their 1865 blood [14]. (ketonemia) These andthree urine compounds (ketourea) based are on collectively known as research done by Gerhardt in 1865 [14]. These three compounds are collectively known as thethe ketone bodies (Figure (Figure1). 1).

FigureFigure 1. The1. The Ketone Ketone Bodies. Bodies. This increase in blood concentrations of the ketone bodies had been shown by Geel- mudyenThis in 1897 increase to be caused in blood by an increase concentrations in the of the of fattyketone acids, bodies due to diabetic had been shown by Geel- patients’mudyen inability in 1897 to metabolize to be caused by at an a rate increase fast enough in the to meet metabolism caloric needs of [15 fatty]. acids, due to dia- This can be dangerous for those with diabetes because unchecked ketonemia can lead tobetic a state patients’ we now know inability as diabetic to metabolize , whereglucose excess at ketonea rate bodies fast enough acidify the to meet caloric needs blood,[15]. This leading can to be nausea, dangerous weakness, for and those even death.with di Woodyattabetes suggestedbecause aunchecked diet based ketonemia can lead onto Newburgha state we and now Marsh’s know research as diabetic where the ketoacid amount ofosis, where excess consumed ketone did bodies acidify the not exceed the quantity the body could metabolize, and to supplement the diet with fats. Thisblood, prevented leading the to buildup nausea, of sugar weakness, in the blood and (hyperglycemia), even death. oneWoodyatt of the causes suggested of a diet based on ketonemia.Newburgh Woodyatt and Marsh’s believed thatresearch over time, where resting the the am pancreasount of would carbohydrates increase the consumed did not body’sexceed capability the quantity to metabolize the body carbohydrates could metabolize, and a normal diet and could to supplement be resumed. The the diet with fats. This discovery of in 1921 and its use to treat diabetes mellitus starting in 1922 decreased theprevented need for other the treatmentbuildup options, of sugar but in the the interest blood in high-fat, (hyperglycemia), low-carbohydrate one diets of the causes of ketone- continuedmia. Woodyatt to grow [16 believed]. that over time, resting the pancreas would increase the body’s capabilityLater that to same metabolize year, Wilder carbohydrates published a report and based a on normal Geyelin’s diet use ofcould fasting be as aresumed. The discov- treatment for epilepsy and suggested that it may be due to the patients’ ketonemia [17]. Referencingery of insulin Shaffer’s in work,1921 Wilder and its suggested use to inducing treat diabetes ketonemia notmellitus through starting fasting, but in 1922 decreased the throughneed for a high-fat, other low-carbohydratetreatment options, diet he but referred the to inte as therest “ketogenic in high-fat, diet”. Peterman,low-carbohydrate diets con- onetinued of his to colleagues, grow [16]. formulated the optimized amounts of macronutrients for the diet, as being no more than 15 g of carbohydrates a day, 1 g of per kilogram of body weight,Later and the that remaining same caloricyear, Wilder deficit made published up of fats. a This report is nearly based identical on Geyelin’s to the use of fasting as moderna treatment ketogenic for diet, epilepsy a 4:1 ratio and of fatssuggested to carbohydrates that it and may protein be due with to amaximum the patients’ ketonemia [17]. allowanceReferencing of 50 gShaffer’s of carbohydrates work, per Wilder day. Wilder suggested released a inducing report shortly ketonemia after this on not through fasting, three of his patients who saw a sharp decrease in the rate of seizures [18]. but Furtherthrough studies a high-fat, over the nextlow-carbohydrate few years corroborated diet the he use referred of the ketogenic to as the diet “ketogenic diet”. Pe- asterman, a treatment one for of epilepsy his colleagues, [19]. Peterman formulated reported his ownthe observationsoptimized inamounts 1924 across of macronutrients for twothe studiesdiet, as on being the use no of themore ketogenic than 15 diet g compared of carboh toydrates a variety ofa day, other treatments.1 g of protein per kilogram of In the first, nine out of 13 patients using only the ketogenic diet as treatment for their epilepsybody weight, were free and of seizures the remaining for up to a year caloric [1]. In deficit the second made study, up 19 of out fats. of 37 This were is nearly identical to seizure-freethe modern for upketogenic to two years diet, [20 ].a 4:1 In 1927, ratio Talbot of fats reported to carbohydrates a study of 200 children and protein with a maxi- wheremum the allowance ketogenic diet of provided50 g of complete carbohydrates symptomatic per relief da amongy. Wilder a third released and partial a report shortly after this on three of his patients who saw a sharp decrease in the rate of seizures [18]. Further studies over the next few years corroborated the use of the ketogenic diet as a treatment for epilepsy [19]. Peterman reported his own observations in 1924 across two studies on the use of the ketogenic diet compared to a variety of other treatments. In the first, nine out of 13 patients using only the ketogenic diet as treatment for their epilepsy were free of seizures for up to a year [1]. In the second study, 19 out of 37 were seizure- free for up to two years [20]. In 1927, Talbot reported a study of 200 children where the

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ketogenic diet provided complete symptomatic relief among a third and partial improve- Int. J. Mol. Sci. 2021, 22, 5230 3 of 18 ment to three quarters [21]. Helmholz reported the use of the diet across 144 epileptic patients. Forty-six patients were seizure-free, and 34 saw a decrease in the rates of seizure occurrence [22]. improvement to three quarters [21]. Helmholz reported the use of the diet across 144 epilepticThe patients. use Forty-sixof medication patients wereto treat seizure-free, epilepsy and was 34 saw gaining a decrease popularity in the rates around of this time as seizurewell occurrence(Figure 2). [22 Phenobarbital]. was brought to market as an anti-convulsant in 1912 and wasThe one use of of the medication treatment to treat options epilepsy Peterman was gaining compared popularity to around the ketogenic this time asdiet [1,23]. Pheny- welltoin (Figure was2 ).synthesized Phenobarbital in was 1908 brought and to shown market to as anbe anti-convulsant useful for preventing in 1912 and seizures was in 1936 [24]. oneCarbamazepine of the treatment optionsand sodium Peterman compared were to the ketogenicboth brought diet [1 ,to23 ].market Phenytoin in 1962 [25,26]. All was synthesized in 1908 and shown to be useful for preventing seizures in 1936 [24]. Carbamazepineare still used and and sodium are currently valproate on were the both World brought Health to market Organization in 1962 [25, 26Model]. All List of Essential areMedicines. still used and The are emergence currently on theof novel World Healthanticonvul Organizationsant medications Model List of caused Essential the ketogenic diet Medicines.to decrease The in emergence popularity of novel as a anticonvulsanttreatment opti medicationson, possibly caused because the ketogenic it was thought that me- dietdicinal to decrease options in popularitywould eliminate as a treatment behavioral option, ch possiblyanges becausealtogether it was [27]. thought Nonetheless, the ke- that medicinal options would eliminate behavioral changes altogether [27]. Nonetheless, thetogenic ketogenic diet diet never never completely completely disappeared. disappeared. The The difficulty difficulty in developing in developing a widely a widely accepted acceptedmedicinal medicinal treatment treatment protocol protocol to to control control epileptic epileptic seizures seizures led to led its sporadicto its sporadic use. use.

FigureFigure 2. Common 2. Common Anticonvulsant Anticonvulsant Medications. Medications. Johns Hopkins Hospital in particular seemed to use the diet for epilepsy treatment more oftenJohns than Hopkins most health Hospital care centers, in particular publishing reviews seemed and to reports use the on itsdiet efficacy for epilepsy in treatment themore 1980s often and 1990s than [27 most–29]. Thehealth cause care of the centers, diet’s subsequent publishing upswing reviews in popularity and reports is on its efficacy atin least the partially 1980s dueand to 1990s the efforts [27–29]. of Jim The Abrahams, cause of a filmthe directordiet’s subsequent whose son Charlie’s upswing in popularity seizures were treated successfully at Johns Hopkins using the ketogenic diet. His son’s is at least partially due to the efforts of Jim Abrahams, a film director whose son Charlie’s story was documented on Dateline NBC in 1994 and he started a foundation called the Charlieseizures Foundation were treated to spread successfully awareness and at fund Johns research Hopkins into the using ketogenic the diet ketogenic [30]. It diet. His son’s is likelystory becausewas documented of this increase on in Dateline publicity that NBC popularity in 1994 and and interest he started in the diet a foundation grew, called the leadingCharlie to a Foundation surge of research to inspread the past awareness twenty-five and years fund [31]. This research research into has the helped ketogenic to diet [30]. It expandis likely the benefitsbecause of of the this ketogenic increase diet beyondin publicity its use that as a treatmentpopularity for epilepsyand interest [32]. in the diet grew, While early use of the ketogenic diet to treat epilepsy was done without a full un- derstandingleading to of a the surge biochemistry of research through in whichthe past ketone twenty-five bodies are generatedyears [31]. and This utilized, research has helped researchto expand over the the past benefits century of has the further ketogenic elucidated diet these beyond pathways. its use as a treatment for epilepsy [32]. While early use of the ketogenic diet to treat epilepsy was done without a full under- 3. Competing Energy Sources in the Body standing of the biochemistry through which ketone bodies are generated and utilized, Energy for the body comes from the digestion and metabolic breakdown of macronu- trientsresearch (fats, carbohydrates,over the past andcentury protein) has in further the diet. elucidated The body prioritizes these pathways. carbohydrate

3. Competing Energy Sources in the Body Energy for the body comes from the digestion and metabolic breakdown of macro- nutrients (fats, carbohydrates, and protein) in the diet. The body prioritizes due to their availability from the diet and from stored in the

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and in muscles, chemically converting them through to glucose, which is oxi- Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 18 Int. J.datively Mol. Sci. 2021 broken, 22, 5230 down through to S-acetyl (or S-acetyl CoA).4 of S- 18 Acetyl CoA is needed to fuel the Krebs cycle, which completes the conversion of upstream carbon sources to and (ATP), the primary source of energy in cells (Figuremetabolismand in 3). muscles, due to their chemically availability converting from the diet them and from through stored hydrolysis glycogen in the to liver glucose, and which is oxi- indatively muscles, broken chemically down converting through them throughglycolysis hydrolysis to S-acetyl to glucose, coenzyme which is oxidativelyA (or S-acetyl CoA). S- brokenAcetyl down CoA through is needed glycolysis to fuel to the S-acetyl Krebs coenzyme cycle, which A (or S-acetyl completes CoA). the S-Acetyl conversion CoA of upstream is needed to fuel the Krebs cycle, which completes the conversion of upstream carbon sourcescarbon to sources carbon dioxide to carbon and adenosine dioxide triphosphateand adenos (ATP),ine triphosphate the primary source (ATP), of energy the primary source inof cellsenergy (Figure in3 cells). (Figure 3).

Figure 3. Adenosine Triphosphate (ATP).

Fats can likewise be broken down hydrolytically to fatty acids that metabolically pro- duce S-acetyl CoA forFigureFigure the 3.Krebs 3.Adenosine Adenosine cycle. Triphosphate ThisTriphosphate occurs (ATP). through(ATP). a repetitive β-oxidation process in the ofFats liver can cells likewise (mostly) be broken. Normally, down hydrolytically these two to fatty pathways acids that metabolicallyto S-acetyl Fats can likewise be broken down hydrolytically to fatty acids that metabolically pro- CoA are complementaryproduce and S-acetyl carefully CoA for regulated the Krebs cycle. metabolically. This occurs through However, a repetitive whenβ-oxidation pushed processduce S-acetyl in the mitochondrion CoA for the Krebs of liver cycle. cells (mostly). This occurs Normally, through these a tworepetitive pathways β-oxidation to process beyond this regulation by dietary consumption or by the body’s immediate energy needs, S-acetylin the mitochondrion CoA are complementary of liver and cells carefully (mostly) regulated. Normally, metabolically. these However, two pathways when to S-acetyl the pathways can becomepushedCoA are beyond competitive. complementary this regulation Indeed byand dietary, carefullyby consumptionlimiting regulated the or byavailability metabolically. the body’s immediate of carbohy-However, energy when pushed drates in the diet andneeds, the theaccumulation pathways can becomeof stored competitive. glucose Indeed,(glycogen by limiting reserves), the availability the body of carbohydratesbeyond this inregulation the diet and by the dietary accumulation consumptio of storedn glucoseor by the (glycogen body’s reserves), immediate the energy needs, can switch to the usebodythe of dietarypathways can switch or to canstored the usebecome offats dietary tocompetitive. produce or stored fats the Indeed to S-acetyl produce, by the limitingCoA S-acetyl needed CoAthe neededavailability for the for of carbohy- Krebs cycle (Figure 4).thedrates Krebs in cycle the (Figurediet and4). the accumulation of stored glucose (glycogen reserves), the body can switch to the use of dietary or stored fats to produce the S-acetyl CoA needed for the Krebs cycle (Figure 4).

Figure 4. Figure 4. Metabolic Sources of S-AcetylMetabolic CoA. Sources of S-Acetyl CoA.

Figure 4. Metabolic Sources of S-Acetyl CoA. 4. Dietary Fats 4. Dietary Fats While the ratio of macronutrients in the ketogenic diet has remained relatively un- While the ratio changed4.of Dietary macronutrients since Fats its development in the in ke 1921,togenic there isdiet some has variety remained within the relatively dietary fats un- we changed since its developmentconsume.While Fats the arein lipophilic,1921,ratio of there macronutrients largely is water-insolublesome variety in the substances withinketogenic that the candiet dietary be has extracted remained fats from we relatively un- living cells with common organic solvents, such as hexane or , and depending consume. Fats are lipophilic,onchanged their structures, largelysince its canwater-inso development appear as oils,luble semi-solids,in substances1921, there or greasy thatis some waxescan varietybe at roomextracted within temperature. from the dietary fats we living cells with commonCollectively,consume. organic Fats these solvents, are organic-soluble lipophilic, such largely compoundsas hexane water-inso canor bediethyl referredluble ether, substances to commonly and depending that as fats, can and be extracted from on their structures, canincludeliving appear cells a wide withas range oils, common of semi-solids, structural organic types orsolvents, that greasy include such waxes long as chain hexane at esterroom derivatives,or temperature.diethyl suchether, as and depending Collectively, these organic-solubletriacylglycerides,on their structures, branched compounds can chain appear compounds can as beoils, referred comprised semi-solids, to of acommonly wideor greasy assortment waxes as offats, terpenes at androom temperature. and terpenoids, such as the (e.g., ) and their fatty esters. Figure5 include a wide rangeillustratesCollectively, of structural some these examples types organic-soluble that of common include dietary longcompounds . chain estercan be derivatives, referred to suchcommonly as as fats, and triacylglycerides, branchedinclude chain a wide compounds range of structural comprised types of that a wide include assortment long chain of ester terpenes derivatives, such as and terpenoids, suchtriacylglycerides, as the steroids (eg., branched cholesterol) chain compounds and their cofattymprised esters. of Figurea wide assortment5 illus- of terpenes trates some examplesand of commonterpenoids, dietary such as lipids. the ster oids (eg., cholesterol) and their fatty esters. Figure 5 illus- trates some examples of common dietary lipids.

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FigureFigure 5. Examples 5.Figure Examples 5. ofExamples of Common Common of DietaryDietary Common Fats.Fats. Dietary Fats.

DietaryDietary fats fats are are primarily primarily triacylglycerides (), (triglycerides), a a glycerol backbone backbone bound bound to threethreeto three carboxyliccarboxylic carboxylic acids acids acids with withwith long long aliphatic aliphaticaliphatic chains chains chains (fatty(fatty (fatty acids) acids) via via viaester ester ester bonds. bonds. bonds. Mono- Monoacyl- Mono- acylglycerides and diacylglycerides are also naturally occurring, but not as abundant in glyceridesacylglycerides and and diacylglycerides diacylglycerides are alsoare also naturally naturally occurring, occurring, but notbut asnot abundant as abundant in the in the diet (Figure 6). dietthe diet (Figure (Figure6). 6).

Figure 6. Dietary Acylglyceride Fats.

The triacylglycerides found in the diet are composed of three long-chain fatty acids Figure 6. Dietary Acylglyceride Fats. Figure(between 6. Dietary 14 and Acylglyceride 22 carbons) Fats.and may be identical or different in the carbon chain length or in the presence of unsaturation or oxygenation in the chain [33]. The number of alkenes The triacylglycerides found in the diet are composed of three long-chain fatty acids inThe the chain triacylglycerides can vary as well, found from in being the satudietrated, are composed monounsaturated, of three or long-chain polyunsaturated. fatty acids (between(betweenThe alkene 1414 andand geometry 2222 carbons) carbons) can likewise and and may maybe either be be identical identi E or Z,cal although or or different different nearly in in theall the examples carbon carbon chain in chain nature length length or inor theinare the presenceZ. presenceThe length of unsaturationof of unsaturation the carbon orchain, oxygenationor oxygenation and the location in thein the or chain the chain type [33 ].[33]. of The alkene(s) The number number within of alkenesof the alkenes in thein thefatty chain chain ester can can chain, vary vary largely as as well, well, determine from from being being the physic saturated, saturated,al and monounsaturated, tomonounsaturated, some extent the biochemical oror polyunsaturated.polyunsaturated. prop- Theerties alkene of the geometry , can likewisewith fully besaturate eitherd EEchains or Z, or although those having nearly E-alkenes all examples being more inin nature are conformationally-rigidZ. The The length length of of the the carbonand carbon leading chain, chain, to compoundsand and the the location locationwith higher or orthe melting the type type of points. alkene(s) of alkene(s) Plants within and within the thefattyanimals fatty ester ester chain,that chain,live largely in cold largely determine environments determine the producephysic the physicalal a andgreater to and someabundance to someextent of extent the triglycerides biochemical the biochemical and prop- propertiesertiesother of thefatty of triglyceride, thelipids triglyceride, that remain with withfullyfluid at fullysaturate low saturated temperatures,d chains chains or depending those or thosehaving on having the E-alkenes required E-alkenes being func- beingmore tion and physical state. moreconformationally-rigid conformationally-rigid and leading and leading to compounds to compounds with withhigher higher melting melting points. points. Plants Plants and and animalsThe fatty that acid live inconcentrations cold environments in triacylg producelycerides a greateralso vary abundance greatly based of triglycerides on the animalssource that of fat. live The in concentrationscold environments of specific produce fatty aacids greater in different abundance sources of oftriglycerides fat can be and and other fatty lipids that remain fluid at low temperatures, depending on the required otheranalyzed fatty lipids by cleaving that remain the fatty fluid acids at from low triacylglycerides temperatures, anddepending converting on them the required into their func- functiontion and andphysical physical state. state. The fattyfatty acidacid concentrations concentrations in triacylglyceridesin triacylglycerides also also vary vary greatly greatly based based on the on source the of fat. The concentrations of specific fatty acids in different sources of fat can be analyzed source of fat. The concentrations of specific fatty acids in different sources of fat can be byanalyzed cleaving by thecleaving fatty the acids fatty from acids triacylglycerides from triacylglycerides and converting and converting them intothem their into estertheir forms. Using these methods, the primary fatty acids in different fats has been studied (Figure7)[33,34].

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Int. J. Mol. Sci. 2021ester, 22, 5230 forms. Usingester these forms. methods, Using the these primary methods, fatty acids the primary in different fatty fats acids has been in different studied fats6 of 18 has been studied (Figure 7) [33,34]. (Figure 7) [33,34].

Figure 7. Major Fatty Acids in Common Foods.

These concentrationsFigureFigure 7. Major7. Major can Fatty Fattyalso Acids beAcids in Commonaffected in Common Foods.by environmental Foods. factors within specific dietary fat sources [35]. These concentrations can also be affected by environmental factors within specific The long dietaryalkyl chainsThese fat sources foundconcentrations [35 in]. most dietary can also fats arebe usefulaffected biofuels by environmental for cells, as their factors within specific carbons are in dietarya higherThe long fat reduction sources alkyl chains state[35]. found (typically in most -2 dietary or -3) fats than are those useful in biofuels carbohydrates for cells, as their (around 0). Consequently,carbonsThe are long in fats a higheralkyl store reductionchains about twfound stateice the (typicallyin amountmost dietary -2 of or potential -3) thanfats are those energy useful in carbohydrates within biofuels for cells, as their their structures,(aroundcarbons which 0). is are Consequently,released in a higherwhen fats converted reduction store about in statecells twice to the(typically CO amount2. of-2 potentialor -3) than energy those within in carbohydrates their structures, which is released when converted in cells to CO . (around 0). Consequently, fats store about twice the2 amount of potential energy within 5. Biochemistry of the Ketogenic Diet 5.their Biochemistry structures, of the which Ketogenic is released Diet when converted in cells to CO2. The biochemistry behind the ketogenic diet is relatively simple. The ketogenic diet The biochemistry behind the ketogenic diet is relatively simple. The ketogenic diet attempts to emulateattempts5. Biochemistry the to body’s emulate response of the the body’s Ketogenic to responsestarvation Diet to or fasting by or fastingeliminating by eliminating carbohy- carbo- drates as the providerhydrates asof theS-acetyl provider CoA, of and S-acetyl thus CoA, the andbody´s thus source the body of ´energy.s source When of energy. car- When bohydrates arecarbohydrates unrestrictedThe biochemistry arein unrestrictedthe diet, behindthe in gl theucose the diet, presentketogenic the glucose in cells diet present or is in relatively in the cells blood or in simple. is the en- blood The is ketogenic diet zymatically convertedenzymaticallyattempts to topyruvate convertedemulate thro tothe pyruvateugh body’s the glycolytic through response the pathway glycolytic to starvation (Figure pathway or 8). (Figurefasting 8). by eliminating carbohy- drates as the provider of S-acetyl CoA, and thus the body´s source of energy. When car- bohydrates are unrestricted in the diet, the glucose present in cells or in the blood is en- zymatically converted to pyruvate through the glycolytic pathway (Figure 8).

Figure 8. Metabolism of Glucose.Figure 8. Metabolism of Glucose.

The glycolysisThe pathway glycolysis generates pathway generates energy energyin the inform the formof adenosine of adenosine triphosphate triphosphate (ATP). The pyruvate molecule is then converted to S-acetyl CoA to be used as fuel for the Krebs (ATP). The pyruvate molecule is then converted to S-acetyl CoA to be used as fuel for the cycle, the body’s primary process for generating energy in the form of ATP (and its direct Krebs cycle, thelinkFigure body’s to the 8. primary mitochondrialMetabolism process of electron-transport Glucose. for gene rating chain). energy in the form of ATP (and its direct link to the mitochondrialWhen carbohydrates electron-transport are abundantly chain). available in the food we eat, the increase in When carbohydratesblood glucoseThe glycolysis are concentration abundantly pathway signals availabl thegeneratese pancreas in the energyfood to secrete we in eat, insulin, the the form aincrease protein of adenosine hormonein triphosphate blood glucosethat (ATP).concentration stimulates The pyruvate thesignals cellular themolecule absorption pancre asis then ofto glucosesecrete converted frominsulin, the to a blood.S-acetyl protein Insulin-dependent CoAhormone to be used as fuel for the that stimulatesglucoseKrebs the cellular uptakecycle, absorption the is largely body’s responsible of primary glucose for fromprocess excess the glucoseforblood. gene storageInsulin-dependentrating in energy the liver in and glu- the skeletal form of ATP (and its muscle as glycogen, and for the conversion of glucose to glycerol (along with glycerol cose uptake is largelydirect linkresponsible to the mitochondrial for excess glucose electron-transport storage in the liver chain). and skeletal mus- cle as glycogen,from and the for hydrolysis the conversion of triglycerides) of glucose to beto usedglycerol in the (along biosynthesis with glycerol of triacylglycerides from that endWhen up being carbohydrates stored in adipose are abundantly tissue [36]. As availabl a resulte of in these the processes, food we blood eat, the increase in the hydrolysis of triglycerides) to be used in the biosynthesis of triacylglycerides that end glucose concentration is maintained at a steady state of around 100–140 mg/dL of blood, up being storedblood in adipose glucose tissue concentration [36]. As a result signals of these the procespancreses,as bloodto secrete glucose insulin, con- a protein centration is maintainedthat stimulates at a steady the cellular state of absorptionaround 100–140 of glucose mg/dL fromof blood, the blood.and upon Insulin-dependent glu- cose uptake is largely responsible for excess glucose storage in the liver and skeletal mus- cle as glycogen, and for the conversion of glucose to glycerol (along with glycerol from the hydrolysis of triglycerides) to be used in the biosynthesis of triacylglycerides that end up being stored in [36]. As a result of these processes, blood glucose con- centration is maintained at a steady state of around 100–140 mg/dL of blood, and upon

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theand burning upon the of burningglucose ofthrough glucose the through Krebs thecycl Krebse, ultimately cycle, ultimately decreases decreases to a point to where a point glucagonwhere (another (another protein proteinhormone) hormone) is releas ised released by the pancreas by the pancreas to orchestrate to orchestrate the break- the downbreak-down of stored of glycogen stored glycogen to release to releasemore glucose more glucose into the into blood. the blood.These glycogen These glycogen stores typicallystores typically last for last about for abouta day.a When day. When more morecarbohydrates carbohydrates are consumed, are consumed, more more insulin insulin is releasedis released to accelerate to accelerate the the uptake uptake of ofglucose glucose to toreplenish replenish glycogen glycogen stores, stores, and and the the process process repeatsrepeats itself itself [37]. [37 ].An An average average 180-pound 180-pound man man typically typically stores stores around around one pound one pound of gly- of cogenglycogen in the in liver the (giving liver (giving about abouta day’s a worth day’s of worth energy) of energy) but around but 40 around times 40that times amount that inamount fat deposited in fat deposited inside of inside of adipocytes (fat cells). (fat cells). However,However, if if glycogen glycogen stores stores are are depleted depleted and and no no carbohydrates carbohydrates are are consumed, consumed, the the bodybody will will begin begin to to break break down down both both dietary dietary triacylglycerides triacylglycerides and and triacylglycerides triacylglycerides con- con- tainedtained in in adipose adipose cells cells through through the the process process of of lipolysis (Figure (Figure 99))[ [38].38]. The The first first step step in in lipolysislipolysis is is the the removal removal of of the the first first fatty , acid, in in this this case case stearic stearic acid, acid, from from the the triglyceride. triglyceride. ItIt requires requires a a unique unique lipase lipase called called adipose adipose triglyceride triglyceride lipase. lipase. Although Although there there are are three di- dia- acylglycerolcylglycerol stereoisomers, adipose triglyceri triglyceridede lipase lipase shows shows a a preference preference for for hydrolyzing hydrolyzing thethe ester ester at at the the sn-2 sn-2 position position [39]. [39]. Similarly, Similarly, hormone-sensitive hormone-sensitive lipase lipase hydrolyzes hydrolyzes diacyl- diacyl- glycerolglycerol with with a apreference preference for for 1,3-diacylglyc 1,3-diacylglycerols.erols. Monoacylglycerol Monoacylglycerol lipase lipase hydrolyzes hydrolyzes the the finalfinal fatty fatty acid acid to to generate generate glycerol glycerol and and a a ne nett total total of of three three fatty fatty acids. acids. These These enzymes enzymes are are relativelyrelatively nonspecific nonspecific because because of of the the large large variety variety of of unique unique triacylglcerides triacylglcerides in in the the diet. diet.

Figure 9. LipolysisFigure 9.ofLipolysis Stored Triacylglycerides. of Stored Triacylglycerides.

DietaryDietary fats fats are are broken broken down down by by a a simila similarr method. method. Lingual Lingual lipases lipases secreted secreted in in the the mouthmouth and and gastric gastric lipases lipases in in the the stomach stomach break break down down some some triacylglycerides triacylglycerides into into mono- mono- andand diacylglycerides. diacylglycerides. Bile Bile salts salts in in the the small small intestine intestine emulsify emulsify fats fats and and allow allow pancreatic pancreatic lipaselipase to to do do thethe same.same. FreeFree fatty fatty acids acids and and the the mono- mono- and and diacylglycerides diacylglycerides are thenare then absorbed ab- sorbedby intestinal by intestinal mucosal mucosal cells and cells combined and combined to form to triacylglycerides form triacylglycerides that can that be dissolvedcan be dis- in solvedlipoprotein in lipoprotein complexes complexes called chylomicrons. called chylom Chylomicronsicrons. Chylomicrons are able to are carry able insoluble to carry fatsin- solublethrough fats the through bloodstream. the bloodstream. They are absorbed They are by liver,absorbed adipose, by liver, and muscleadipose, cells and where muscle the cellstriacylglycerides where the triacylglycerides are hydrolyzed byare lipoprotein hydrolyzed and by hepaticlipoprotein triglyceride and hepatic lipases triglyceride to generate lipasesfree fatty to generate acids. free fatty acids. The fatty acids released either through lipolysis or through the digestion of dietary The fatty acids released either through lipolysis or through the digestion of dietary fats are carried through the blood by albumin until they are absorbed by cells and used as fats are carried through the blood by albumin until they are absorbed by cells and used as an energy source. The glycerol released by the breakdown of triacylglcyerides can be used an energy source. The glycerol released by the breakdown of triacylglcyerides can be used

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to synthesizeto synthesize new new triacylglycerides triacylglycerides or or can can be be converted converted in the in liverthe liver to D-glyceraldehyde-3- to D-glyceraldehyde- 3-phosphatephosphate and enterenter thethe gluconeogenesis pathway pathwa toy generateto generate glucose, glucose, but thisbut willthis not will not fullyfully meet meet the the caloric caloric requirements requirements ofof the the body body (Figure (Figure 10 ).10).

Figure 10.Figure Metabolic 10. Metabolic Conversion Conversion of Glycerol of Glycerol to Glucose. to Glucose.

Instead, the fatty acids released by lipolysis are absorbed by the cells and converted Instead, the fatty acids released by lipolysis are absorbed by the cells and converted into S-acetyl CoA through ββ-oxidation. This process is a repeating set of steps that converts Int. J. Mol. Sci. 2021, 22, x FOR PEERinto theREVIEW S-acetylβ-carbon CoA of the through fatty acid -oxidation. into a carbonyl This group process and is then a repeating cleaves off set an S-acetylof steps CoA,9 that of 18 con- β vertsshortening the -carbon the fatty of acidthe fatty (Figure acid 11 ).into a carbonyl group and then cleaves off an S-acetyl CoA, shortening the fatty acid (Figure 11). The first step in fatty acid β-oxidation is the thioesterification by coenzyme A, which assists in transport to the mitochondrial outer membrane. Inside the mitochondrion, the first step is a regioselective dehydrogenation reaction. Next, the alkene is hydrated to gen- erate a hydroxyl group at the β-carbon. This addition is stereoselective for the S-enantio- mer. The penultimate step is the NAD+ oxidation of the secondary to a ketone. Finally, S-acetyl CoA is cleaved, and the resulting fatty acid chain is two carbon atoms shorter. The cycle repeats itself until the fatty acid is fully converted to S-acetyl CoA with some minor differences for unsaturated fatty acids and those with odd-numbered carbon chain lengths.

FigureFigure 11.11. β-Oxidation of a Fatty Acid.

The S-acetyl CoA generated by cells through β-oxidation can directly enter the Krebs cycle and provide energy in the form of ATP. For most of the cells in the body, this process could supply their energy needs until triacylglyceride stores are depleted. However, be- cause fatty acids cannot cross the blood– barrier, they cannot be absorbed and used by cells in the brain. The brain uses approximately 20% of the energy the body requires, which necessitates an effective method for providing lasting energy when carbohydrate consumption is low [3,40]. The body overcomes this obstacle by converting S-acetyl CoA into the three ketone bodies (Figure 1), water-soluble compounds that can enter the blood, cross the blood– brain barrier, and be absorbed by neuronal cells in the brain before being converted to S- acetyl CoA to fuel the Krebs cycle. While they are presented here in their acid forms, these two acids are deprotonated at physiological pH and referred to as acetoacetate and (R)-β- hydroxybutyrate [41]. The metabolic process of generating ketone bodies from S-acetyl CoA occurs through ketogenesis, and takes place primarily in the liver (Figure 12). The first step is the Claisen- type condensation of two molecules of S-acetyl CoA to form S-acetoacetyl CoA. This is followed by an aldol addition of another S-acetyl CoA to the β-carbonyl with subsequent hydrolysis of one of the coenzyme A thioesters to generate a . The S-acetyl CoA is cleaved by HMG-CoA lyase to generate , which can subsequently be reduced by 3-hydroxybutyrate dehydrogenase to generate (R)-β-hydroxybutyric acid. Decarboxylation of acetoacetic acid, as a means to release acetone and carbon dioxide, can occur spontaneously as well, although this is primarily the method through which excess ketone bodies are removed as waste.

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The first step in fatty acid β-oxidation is the thioesterification by coenzyme A, which assists in transport to the mitochondrial outer membrane. Inside the mitochondrion, the first step is a regioselective dehydrogenation reaction. Next, the alkene is hydrated to generate a hydroxyl group at the β-carbon. This addition is stereoselective for the S- enantiomer. The penultimate step is the NAD+ oxidation of the secondary alcohol to a ketone. Finally, S-acetyl CoA is cleaved, and the resulting fatty acid chain is two carbon atoms shorter. The cycle repeats itself until the fatty acid is fully converted to S-acetyl CoA with some minor differences for unsaturated fatty acids and those with odd-numbered carbon chain lengths. The S-acetyl CoA generated by cells through β-oxidation can directly enter the Krebs cycle and provide energy in the form of ATP. For most of the cells in the body, this process could supply their energy needs until triacylglyceride stores are depleted. However, because fatty acids cannot cross the blood–brain barrier, they cannot be absorbed and used by cells in the brain. The brain uses approximately 20% of the energy the body requires, which necessitates an effective method for providing lasting energy when carbohydrate consumption is low [3,40]. The body overcomes this obstacle by converting S-acetyl CoA into the three ketone bodies (Figure1), water-soluble compounds that can enter the blood, cross the blood– brain barrier, and be absorbed by neuronal cells in the brain before being converted to S-acetyl CoA to fuel the Krebs cycle. While they are presented here in their acid forms, these two acids are deprotonated at physiological pH and referred to as acetoacetate and (R)-β-hydroxybutyrate [41]. The metabolic process of generating ketone bodies from S-acetyl CoA occurs through ketogenesis, and takes place primarily in the liver (Figure 12). The first step is the Claisen- type condensation of two molecules of S-acetyl CoA to form S-acetoacetyl CoA. This is followed by an aldol addition of another S-acetyl CoA to the β-carbonyl with subsequent hydrolysis of one of the coenzyme A thioesters to generate a carboxylic acid. The S-acetyl CoA is cleaved by HMG-CoA lyase to generate acetoacetic acid, which can subsequently be reduced by 3-hydroxybutyrate dehydrogenase to generate (R)-β-hydroxybutyric acid. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWDecarboxylation of acetoacetic acid, as a means to release acetone and carbon dioxide, can10 of 18 occur spontaneously as well, although this is primarily the method through which excess ketone bodies are removed as waste.

S-AcCoA CoA-SH H O CoA-SH O OO 2 O HO O

S-CoA S-CoA HMG-CoA HO S-CoA synthase S-acetyl CoA S-acetoacetyl CoA 3-hydroxy-3-methylglutaryl CoA (2 moles)

HMG-CoA lyase S-AcCoA NAD NAD+ H/H+ OH O OO

OH 3-hydroxybutyrate OH dehydrogenase acetoacetic acid (R)-ß-hydroxybutyric acid FigureFigure 12. Ketogenesis 12. Ketogenesis of Ketone of Ketone Bodies Bodies from from S-Acetyl S-Acetyl CoA. CoA.

While S-acetoacetyl CoA could be hydrolyzed to acetoacetic acid directly, there is While S-acetoacetyl CoA could be hydrolyzed to acetoacetic acid directly, there is evidence that the expression of HMG-CoA synthase plays a role in limiting the rate at evidence that the expression of HMG-CoA synthase plays a role in limiting the rate at which ketogenesis occurs [42]. The ketone bodies produced metabolically in the liver are released into the blood where they can be absorbed by other cells and used to synthesize S-acetyl CoA through the process of ketolysis (Figure 13). First, NAD+ oxidation converts (R)-β-hydroxybutyric acid to acetoacetic acid, which undergoes thioesterification with co- enzyme A to generate S-acetoacetyl CoA. S-Acetoacetyl CoA is cleaved by coenzyme A through a retro-Claisen reaction to afford two S-acetyl CoA molecules that can directly enter the Krebs cycle.

Figure 13. Ketolysis of the Ketone Bodies.

6. Metabolic Effect of Ketolysis Versus Glycolysis Bypassing the traditional pathways of releasing energy through glycolysis in favor of using ketone bodies has a profound effect on the body. While the complete mechanism is not fully understood, bypassing carbohydrate metabolism pathways in the brain can lead to a decreased incidence or even the elimination of epileptic seizures [6]. The state of having elevated ketone bodies in the blood is called ketosis. Typical ke- tone body concentrations during dietary ketosis are between 0.5–3 mM, while a normal

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S-AcCoA CoA-SH H O CoA-SH O OO 2 O HO O

S-CoA S-CoA HMG-CoA HO S-CoA Thiolase synthase S-acetyl CoA S-acetoacetyl CoA 3-hydroxy-3-methylglutaryl CoA (2 moles)

HMG-CoA lyase S-AcCoA NAD NAD+ H/H+ OH O OO

OH 3-hydroxybutyrate OH dehydrogenase acetoacetic acid (R)-ß-hydroxybutyric acid Figure 12. Ketogenesis of Ketone Bodies from S-Acetyl CoA.

Int. J. Mol. Sci. 2021, 22, 5230 While S-acetoacetyl CoA could be hydrolyzed to acetoacetic acid10 of 18directly, there is evidence that the expression of HMG-CoA synthase plays a role in limiting the rate at which ketogenesis occurs [42]. The ketone bodies produced metabolically in the liver are whichreleased ketogenesis into the occurs blood [42 where]. The ketone they bodiescan be produced absorbed metabolically by other cells in the and liver used are to synthesize releasedS-acetyl into CoA the through blood where the they process can be of absorbed ketolysis by other(Figure cells 13). and First, used to NAD synthesize+ oxidation converts + S-acetyl(R)-β-hydroxybutyric CoA through the process acid to of acetoacetic ketolysis (Figure acid, 13 which). First, NADundergoesoxidation thioesterification converts with co- (R)-β-hydroxybutyric acid to acetoacetic acid, which undergoes thioesterification with coenzymeenzyme A toto generate generate S-acetoacetyl S-acetoacetyl CoA. CoA. S-Acetoacetyl S-Acetoacetyl CoA is cleaved CoA byis cleaved coenzyme by coenzyme A Athrough through aa retro-Claisenretro-Claisen reaction reaction to afford to afford two S-acetyl two CoAS-acetyl molecules CoA that molecules can directly that can directly enterenter the the Krebs Krebs cycle. cycle.

FigureFigure 13. 13.Ketolysis Ketolysis of the of Ketone the Ketone Bodies. Bodies. 6. Metabolic Effect of Ketolysis Versus Glycolysis 6. MetabolicBypassing the Effect traditional of Ketolysis pathways Versus of releasing Glycolysis energy through glycolysis in favor of using ketoneBypassing bodies the has traditional a profound effect pathways on the body. of releasing While the energy complete through mechanism glycolysis is in favor notof using fully understood, ketone bodies bypassing has carbohydratea profound metabolismeffect on the pathways body. in While the brain the can complete lead mechanism to a decreased incidence or even the elimination of epileptic seizures [6]. is notThe fully state understood, of having elevated bypassing ketone bodiescarbohydra in the bloodte metabolism is called ketosis. pathways Typical in the brain can ketonelead to body a decreased concentrations incidence duringdietary or even ketosis the elimination are between 0.5–3 of epileptic mM, while seizures a normal [6]. diet willThe give state ketone of bodyhaving concentrations elevated ketone of less than bodies 0.3 mM in [the43]. Achievingblood is called and keeping ketosis. Typical ke- thetone body body in a stateconcentrations of ketosis is the during goal of dietary the ketogenic ketosis diet, are as anybetween carbohydrates 0.5–3 mM, above while a normal an absolute bare minimum will trigger the release of insulin and rapidly decrease the rate of generation of ketone bodies. It is important to note that this is different than , a potentially life- threatening condition that occurs primarily in type I diabetics where unchecked ketogenesis can cause ketone body concentrations to rise to 10mM and beyond, overwhelming the body’s acid-base buffering system and causing the blood to turn acidic [44]. Insulin acts as an inhibitor for lipolysis, β-oxidation, and ketogenesis and prevents the build-up of ketone bodies to unsafe levels during dietary ketosis [43]. Even for those with type 2 diabetes where insulin sensitivity in the cells is decreased, diabetic ketoacidosis is rare since these catabolic processes are very sensitive to insulin. However, type I diabetics are at risk for diabetic ketoacidosis since the hormone to stop the generation of ketone bodies is present in low amounts or absent entirely. Int. J. Mol. Sci. 2021, 22, 5230 11 of 18

7. Benefits of the Ketogenic Diet The use of the ketogenic diet as a treatment option for epilepsy is well-studied, but research has shown that there are other benefits. The most popular is its efficacy as a weight-loss tool. Statistical analysis of fourteen studies comparing weight loss using the ketogenic diet or low-fat diets showed the ketogenic diet caused greater reductions in body weight [45]. The diet has also been shown to be a more effective method for weight loss compared to a normal diet at similar caloric deficits in trials that range in length from three months to two years [46,47]. It is possible that the initial rapid weight loss when initiating the diet comes from the body’s attempts to use glycerol from triglycerides to overcome a lack of dietary carbohydrates, leading to a decrease in adipose fat stores and thus weight. Converting glycerol into glucose through the gluconeogenesis pathway is also a very energy-demanding process, so the body is using excess energy before it begins to adapt to using ketone bodies as a primary energy source. Tangentially related to the benefits of weight loss, the ketogenic diet has been shown to improve and even reverse in those suffering from type 2 diabetes or for those who are at risk of becoming diabetic [48,49]. Increased insulin resistance has been shown to lead to higher conversion of glucose into triglycerides that can lead to disease [50,51]. For obese patients, the ketogenic diet has been shown to decrease total cholesterol and triglyceride concentrations in the blood, which can decrease the risk of cardiovascular and metabolic diseases [52–54]. Alongside the more well-studied benefits of the diet, there is emerging evidence indicating benefits for other diseases and neurological disorders [55–57]. Patients with Parkinson’s disease have shown improved scores on the Unified Parkinson’s Disease Rating Scale after four weeks on a ketogenic diet [58]. The infusion of (R)-β-hydroxybutyric acid was shown to protect against dopaminergic neurodegeneration induced by neurotoxins that mimic the effects of Parkinson’s disease and Alzheimer’s disease [59,60]. Mouse models have been used to show that the ketogenic diet can decrease the concentration of amyloid-β in the brain, a risk factor for developing Alzheimer’s disease [61]. The ketogenic diet has also demonstrated in mice models to slow the degradation of motor neurons due to amyotrophic lateral sclerosis (ALS) [62]. It has long been recognized that cancer cells consume glucose at a much higher rate than normal cells [63,64]. Increased glycolysis promotes excessive proliferation in cancer cells [65]. However, in vitro studies have shown that a lack of glucose can cause apoptosis in malignant cells [66–68]. Certain tumors are unable to utilize ketone bodies as a source of energy due to a decrease in ketolysis enzyme activity [69,70]. The use of the ketogenic diet to effectively “starve” certain types of tumors could be an adjuvant treatment alongside more traditional forms of cancer treatment. In mice, the ketogenic diet has been shown to slow the rate of tumor growth and improve the effects of radiation treatment [71–73]. There are a few published reports of similar outcomes in human patients, but no formal clinical testing has yet been done [74,75]. While much of this evidence is far from conclusive, it shows the wide range of possibilities the diet holds, which justifies and indeed creates opportunities for further research.

8. Limitations of the Ketogenic Diet Most of the long-term effects of the ketogenic diet are based on reports from the children with epilepsy who were treated by it. Hyperlipidemia has been seen in a majority of children treated with the traditional ketogenic diet, although this can be avoided by adjusting the types of fats consumed [76,77]. Kidney stones are seen in approximately 10% of children on the ketogenic diet [78,79]. There is concern that a strict ketogenic diet may affect growth rates, possibly due to protein or overall calorie restrictions [80,81]. There is some evidence that the ketogenic diet can cause osteopenia, a condition where bone mass is lost, possibly due to vitamin D and calcium deficiencies [82,83]. Research into the long-term effects of the ketogenic diet on healthy adults is sparse, but the recent popularity of the diet makes it likely that these studies will be done. Int. J. Mol. Sci. 2021, 22, 5230 12 of 18

One of the more common criticisms of the ketogenic diet is that it is difficult to maintain. Clinical studies from the diet’s earliest use in the 1920s to treat epilepsy and more recent studies on weight loss commonly report patients that were unable to follow the diet [20–23,45–47]. A single meal with too high a proportion of carbohydrates will cause the body to fall out of ketosis and revert back to using carbohydrates as its main energy source [84,85]. Long-term adherence to the diet can be low due to lack of discipline, adverse gastrointestinal effects, or palatability issues. Modified versions of the ketogenic diet have been developed to try and address these concerns. The MCT (medium-chain triglyceride) diet in particular has seen wide use. It focuses on using naturally-occurring triacylglycerides consisting of medium-chain fatty acids (between 6 and 12 carbons) that do not require active transport into cells and are instead absorbed by the liver directly. This increases the rate at which dietary triglycerides can be converted to ketone bodies, but research has shown that a greater percentage of fatty acids in medium-chain triglycerides are metabolized in the liver and can be used to generate ketone bodies compared to longer fatty acids that are partially used in the synthesis of new triacylglycerides to be stored in adipose tissue [86]. Medium-chain triglycerides have been shown to have a greater effect on ketone body concentration compared to long-chain triglycerides [87]. Because of this, the MCT diet allows for a greater amount of carbohydrates and in the diet and medium-chain triglycerides are considered to be more “ketogenic”; however, it still suffers from many of the same drawbacks as the traditional ketogenic diet, including a reliance on a strict dietary regiment. Improving the effectiveness and sustainability of the ketogenic diet using alternative natural sources of fat does not address many of the primary limiting factors of the diet. Preliminary research into the use of synthetic compounds to induce ketosis could better address these factors.

9. Synthetic Ketogenic Compounds as an Alternative Path to Ketosis Originally, it was believed that the benefits of the ketogenic diet were due to the decrease in glucose metabolism; however, more recent studies have shown that some of these benefits are instead caused by an increase in ketone body concentrations in the blood [88,89]. In the past, this distinction was irrelevant since a normal diet will prevent the body from generating ketone bodies at a rate high enough to maintain a state of ketosis. However, three synthetic ketogenic compounds have been shown to increase ketone body Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 13 of 18 concentration in the blood to the point where dietary ketosis can be maintained without a change from a normal diet (Figure 14).

FigureFigure 14. 14.Synthetic Synthetic Ketogenic Ketogenic Compounds. Compounds.

± ± ( (±)-1,3-Butanediol)-1,3-Butanediol acetoacetate acetoacetate diester diester was synthesized was synthesized from ( )-1,3-butanediolfrom (±)-1,3-butanediol and and t-butyl acetoacetate via a transesterification, published in 1995 (Figure 15)[90]. t-butyl acetoacetate via a transesterification, published in 1995 (Figure 15) [90].

OO

O OO tBuOH t-butyl acetoacetate (2 moles) O O O

140oC OH O (±)-1,3-butanediol acetoacetate diester OH

(±)-1,3-butanediol Figure 15. Synthesis of (±)-1,3-Butanediol Acetoacetate Diester.

This compound is metabolically hydrolyzed to generate two equivalents of acetoace- tic acid and (±)-1,3-butanediol, which can be oxidized to generate β-hydroxybutyric acid (Figure 16) [91,92]. The oxidation of the (R)-enantiomer occurs in the liver. (S)-β-Hydroxy- is not naturally occurring, but its metabolism in perfused rat has been shown to generate ketone bodies through an unknown pathway [93].

Figure 16. Oxidation of (R)-β-1,3-Butanediol in the Liver.

Racemic 1,3-butanediol acetoacetate diester was shown to increase ketone body con- centrations in the blood of both a pig and dog. More recent studies in rats indicate it in- duces a rapid increase in (R)-β-hydroxybutyric acid concentration in the blood and a de- crease in blood glucose concentration, indicative of ketosis [94]. Importantly, triglyceride and cholesterol levels did not change to a statistically relevant level over 28 days of daily administration [6]. A similar compound, (R)-3-hydroxybutyl (R)-3-hydroxybutanoate, was synthesized by Clarke and Veech in 2010 [95]. A lipase-mediated transesterification reaction between

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Figure 14. Synthetic Ketogenic Compounds.

(±)-1,3-Butanediol acetoacetate diester was synthesized from (±)-1,3-butanediol and Figuret-butyl 14. acetoacetate Synthetic Ketogenic via a Compounds.transesterification, published in 1995 (Figure 15) [90].

Int. J. Mol. Sci. 2021, 22, 5230 13 of 18 OO(±)-1,3-Butanediol acetoacetate diester was synthesized from (±)-1,3-butanediol and t-butyl acetoacetate via a transesterification, published in 1995 (Figure 15) [90]. O OO OO tBuOH t-butyl acetoacetate (2 moles) O O O O OO tBuOH t-butyl acetoacetate 140oC (2 moles) OH O O O O (±)-1,3-butanediol acetoacetate diester 140oC OH OH O

(±)-1,3-butanediol (±)-1,3-butanediol acetoacetate diester OH Figure 15. Synthesis of (±)-1,3-Butanediol Acetoacetate Diester. (±)-1,3-butanediol FigureFigure This15. 15. Synthesis compound of of ( ±(±)-1,3-Butanediol)-1,3-Butanediol is metabolically Acetoacetate Acetoacetate hydrolyzed Diester. Diester. to generate two equivalents of acetoace- tic acid and (±)-1,3-butanediol, which can be oxidized to generate β-hydroxybutyric acid ThisThis compoundcompound isis metabolicallymetabolically hydrolyzed hydrolyzed to to generate generate two two equivalents equivalents of of ace- acetoace- toacetic(Figure acid 16) and [91,92]. (±)-1,3-butanediol, The oxidation which of the can (R)-enantiomer be oxidized to generate occursβ-hydroxybutyric in the liver. (S)-β-Hydroxy- tic acid and (±)-1,3-butanediol, which can be oxidized to generate β-hydroxybutyric acid acid (Figure 16)[91,92]. The oxidation of the (R)-enantiomer occurs in the liver. (S)-β- (Figurebutyric 16) acid [91,92]. is not The naturally oxidation occurring, of the (R)-enantiomer but its metabolism occurs in inthe perfused liver. (S)- βrat-Hydroxy- livers has been Hydroxybutyricshown to generate acid is ketone not naturally bodies occurring, through but an its unknown metabolism pathway in perfused [93]. rat livers butyrichas been acid shown is not to generate naturally ketone occurring, bodies throughbut its metabolism an unknown in pathway perfused [93]. rat livers has been shown to generate ketone bodies through an unknown pathway [93].

Figure 16. 16.Oxidation Oxidation of (R)- ofβ (R)--1,3-Butanediolβ-1,3-Butanediol in the Liver. in the Liver. Figure 16. Oxidation of (R)-β-1,3-Butanediol in the Liver. Racemic 1,3-butanediol acetoacetate diester was shown to increase ketone body con- centrationsRacemicRacemic in the1,3-butanediol 1,3-butanediol blood of both acetoacetate a acetoacetate pig and dog. diester diester More was recent shownwas studies shown to increase in to rats increase indicateketone ketonebody it con- body con- centrationsinducescentrations a rapid in in the increasethe blood blood in of (R)- ofbothβ both-hydroxybutyric a pig a pigand and dog. aciddog. More concentration More recent recent studies in thestudies in blood rats inindicate and rats a indicate it in- it in- ducesdecreaseduces a a rapid in rapid blood increase increase glucose in concentration, in(R)- (R)-β-hydroxybutyricβ-hydroxybutyric indicative of acid ketosis acidconcentration [94 ].concentration Importantly, in the triglyceride inblood the andblood a de- and a de- creaseandcrease cholesterol in in blood blood levels glucose glucose did concentration, not concentration, change to a statisticallyindicative indicative of relevant ketosis of ketosis level [94]. over Importantly, [94]. 28 days Importantly, of daily triglyceride triglyceride administration [6]. andand cholesterol cholesterol levels levels did did not not change change to a tostatistically a statistically relevant relevant level over level 28 over days 28 of days daily of daily A similar compound, (R)-3-hydroxybutyl (R)-3-hydroxybutanoate, was synthesized administrationbyadministration Clarke and Veech [6]. [6]. in 2010 [95]. A lipase-mediated transesterification reaction between (R)-1,3-butanediolAA similar similar compound, compound, and ethyl (R)-3-hydroxybutanoate (R)-3-hydroxybutyl (R)-3-hydroxybutyl (R)-3-hydroxybutanoa generates (R)-3-hydroxybutanoa a stereospecificte, monoester waste, synthesized was synthesized bythatby ClarkeClarke can be and hydrolyzedand Veech Veech in to in2010 generate 2010 [95]. [95]. (R)-A lipase-mediated Aβ-hydroxybutyric lipase-mediated transesterification acid transesterification and (R)-1,3-butanediol, reaction reaction between between which can be oxidized in the liver to give a second equivalent of (R)-β-hydroxybutyric acid (Figure 17)[96]. It has been shown in adult humans that daily administration of (R)-3-hydroxybutyl

(R)-3-hydroxybutanoate can safely induce ketosis while on a normal diet over 28 days of administration [97,98]. A final set of ketogenic compounds that have been used to induce ketosis are salts generated from racemic β-hydroxybutyrate. Most commonly, these are sodium, potassium, and calcium salts (Figure 18). In studies in rats fed a normal diet and supplemented with these salts, a small increase in ketone body concentration in the blood was observed. Mineral salts of β-hydroxybutyrate can induce ketosis, but the amounts required can lead to negative gastrointestinal effects and issues due to high levels of sodium [99]. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 14 of 18

(R)-1,3-butanediol and ethyl (R)-3-hydroxybutanoate generates a stereospecific monoes- ter that can be hydrolyzed to generate (R)-β-hydroxybutyric acid and (R)-1,3-butanediol, which can be oxidized in the liver to give a second equivalent of (R)-β-hydroxybutyric acid (Figure 17) [96].

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 14 of 18

(R)-1,3-butanediol and ethyl (R)-3-hydroxybutanoate generates a stereospecific monoes- ter that can be hydrolyzed to generate (R)-β-hydroxybutyric acid and (R)-1,3-butanediol, Int. J. Mol. Sci. 2021, 22, 5230 14 of 18 which can be oxidized in the liver to give a second equivalent of (R)-β-hydroxybutyric acid (Figure 17) [96].

Figure 17. Enzymatic Synthesis of (R)-3-hydroxybutyl (R)-3-hydroxybutanoate.

It has been shown in adult humans that daily administration of (R)-3-hydroxybutyl (R)-3-hydroxybutanoate can safely induce ketosis while on a normal diet over 28 days of administration [97,98]. A final set of ketogenic compounds that have been used to induce ketosis are salts generated from racemic β-hydroxybutyrate. Most commonly, these are sodium, potas- sium, and calcium salts (Figure 18). In studies in rats fed a normal diet and supplemented with these salts, a small increase in ketone body concentration in the blood was observed. Mineral salts of β-hydroxybutyrate can induce ketosis, but the amounts required can lead Figure 17. FigureEnzymatic 17. Enzymatic Synthesis Synthesis of (R)-3-hydroxybutyl of (R)-3-hydroxybutyl (R)-3-hydroxybutanoate. (R)-3-hydroxybutanoate. to negative gastrointestinal effects and issues due to high levels of sodium [99]. It has been shown in adult humans that daily administration of (R)-3-hydroxybutyl (R)-3-hydroxybutanoate can safely induce ketosis while on a normal diet over 28 days of administration [97,98]. A final set of ketogenic compounds that have been used to induce ketosis are salts generated from racemic β-hydroxybutyrate. Most commonly, these are sodium, potas- sium, and calcium salts (Figure 18). In studies in rats fed a normal diet and supplemented with these salts,FigureFigure a small 18. 18. βincrease -Hydroxybutyrateβ-Hydroxybutyrate in ketone Mineral body Mineral Salts. concentration Salts. in the blood was observed. Mineral salts of β-hydroxybutyrate can induce ketosis, but the amounts required can lead to negative gastrointestinalTheThe use use of effects of synthetic synthetic and ketogenic issues ketogenic due compounds to compounds high levels to supplement toof supplement sodium a normal [99]. a normal diet has diet not onlyhas not only shown the capability to induce ketosis, but also consequently decreases blood glucose shown the capability to induce ketosis, but also consequently decreases blood glucose concentration without affecting triglyceride or cholesterol levels [6]. (±)-1,3-Butanediol acetoacetateconcentration diester without has been affecting shown totriglyceride suppress seizure or cholesterol activity in ratslevels [100 [6].]. Combining (±)-1,3-Butanediol (±acetoacetate)-1,3-butanediol diester acetoacetate has been diester shown with to hyperbaricsuppress oxygenseizure therapy activity was in shownrats [100]. to slow Combining metastatic(±)-1,3-butanediol cancer growth acetoacetate in mice [5diester]. Like wi theth benefits hyperbaric of the oxygen ketogenic therapy diet itself, was the shown to researchslow metastatic into the usecancer of synthetic growth ketogenicin mice [5]. compounds Like the benefits to supplement of the ketogenic a normal diet diet is itself, the promisingresearch into but isthe in itsuse infancy. of synthetic Similarly, ketogeni the varietyc compounds of synthetic to ketogenicsupplement compounds a normal diet is

availablepromising is currentlybut is in extremely its infancy. limited Similarly, to those the described, variety andof synthetic thus there ketogenic remains enor- compounds Figure 18. β-Hydroxybutyrate Mineral Salts. mousavailable potential is currently to further extremely expand on limited this through to those targeted described, organic and synthesis, thus there to advance remains enor- the breadth of research on ketogenic substances and their use in improving human health. mous potential to further expand on this through targeted organic synthesis, to advance The use of synthetic ketogenic compounds to supplement a normal diet has not only the breadth of research on ketogenic substances and their use in improving human health. shown the capability10. Conclusions to induce ketosis, but also consequently decreases blood glucose concentration withoutFrom affecting a chemist’s triglyceride perspective, or the cholesterol field of synthetic levels ketogenic [6]. (±)-1,3-Butanediol compounds is vast and 10. Conclusions acetoacetate diestervirtually has unexplored. been shown Palatability, to suppress caloric seizure density, activity , in rats and [100]. gastrointestinal Combining tolerance are all attributes that can be improved. Advances in food formulation of emulsions (±)-1,3-butanediol acetoacetateFrom a chemist’s diester perspective, with hyperbaric the field oxygen of synthetic therapy ketogenic was shown compounds to is vast and may be the most obvious immediate options, but there is also a need for a significant slow metastatic virtuallycancer growth unexplored. in mice Palatability,[5]. Like the benefitscaloric density, of the ketogenic solubility, diet and itself, gastrointestinal the toler- expansionance are all of edible,attributes ketogenic that can food be substances improved. whose Advances structures, in food purities, formulation and biochemical of lipid emul- research into theeffects use areof synthetic all well-documented. ketogenic compounds Different functional to supplement groups linking a normal together diet ketogenicis sions may be the most obvious immediate options, but there is also a need for a significant promising but groupsis in its could infancy. lead toSimilarly, a better controlled the variety rate of of synthetic generation ketogenic of ketone bodies,compounds perhaps even available is currentlytissue-specific. extremely The limited possibilities to th areose nearlydescribed, endless and and thus limited there only remains by the enor- imaginative mous potentialabilities to further and expand restrictions on ofthis chemical through synthesis. targeted organic synthesis, to advance the breadth of researchIt is rareon ketogenic to find an areasubstanc of researches and in their the fielduse ofin chemistryimproving that human is mostly health. unexplored. Small-molecule synthesis is of particular interest to organic chemists for pharmaceutical

10. Conclusionsdevelopment across a broad spectrum of human diseases, but whether it is the ketogenic diet’s origins as a medical treatment or the relative recency of much of the research, From a chemist’sthere has perspective, been little effort the field put forth of synthetic to explore ketogenic the field ofcompounds ketogenic compoundsis vast and among virtually unexplored.synthetic Palatability, organic chemists. caloric There density, is both commercialsolubility, andand biomedical gastrointestinal potential toler- to providing ance are all attributesalternative that methods can be improved. of inducing Advances and maintaining in food ketosis, formulation and novel of lipid synthetic emul- ketogenic sions may be thecompounds most obvious could immediate be an important options, aspect but of there a solution. is also a need for a significant

Int. J. Mol. Sci. 2021, 22, 5230 15 of 18

Author Contributions: All authors contributed to the conceptualization, writing, and editing of this manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest.

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