Caprylic (Octanoic) Acid as a Potential Fatty Acid Chemotherapeutic for Glioblastoma

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Highlights

Abstract

High grade glial tumors (HGGs) including anaplastic astrocytoma (WHO Grade-III) and glioblastoma multiforme (GBM, WHO Grade-IV) are among the most malignant cancers known to man. Due to their defective mitochondria, HGG cells consume glucose via glycolysis even in the presence of oxygen. Overall survival is worse in HGG patients that are hyperglycemic. Unlike normal neural cells, HGG cells cannot efficiently metabolize ketone bodies for energy. Thus, a metabolic treatment based on therapeutic ketosis (reduced glucose with elevated ketone bodies) was proposed to treat GBM and was supoported from preclinical studies. Caprylic (octanoic) acid, a monocarboxylated saturated fatty acid, is among the best producers of ketone bodies and induces necrosis of experimental tumors at high dose. Caprylic acid is enriched in coconut and in goat's milk. It is also a posttranslational modifier of the ghrelin hormone and is produced in trace amounts in human tissues. Caprylic acid is a straight-chain isomer of the antiepileptic valproic acid, which is used in treatment of HGG-associated seizures and which may increase survival in GBM patients according to epidemiological observations. Among the valproic acids analogs tested, caprylic acid is the most potent molecule to block C6 astrocytoma cell growth in vitro and accumulates selectively within glial cells as shown by Positron Emission Tomography in vivo. Caprylic acid blocks glycolysis both in healthy liver and in malignant liver cells, which is more prominent in the latter and also lowers blood glucose. Noteworthy, caprylic acid exerts neuroprotective- and mitochondria-protective effects in several models of neurodegenerative diseases. Boost injections of caprylic acid at non-toxic levels during classical ketogenic metabolic therapy may fortify antitumor actions and reduce systemic toxicity by differential programming of mitochondrial and other metabolic pathways.

Introduction

Gliomas are the most frequently encountered human brain tumors and contain histologic subtypes including astrocytomas, oligodendrogliomas and ependymomas [1]. Gliomas are classified as grades I–IV on the basis of pathological criteria established by the World Health Organization (WHO) [1]. While WHO grade I gliomas are mostly treatable with total surgical resection, gliomas of WHO grade II or III are invasive, progress to higher-grade tumors and have a poor prognosis [1]. Glioma WHO Grade-IV or so called Glioblastoma Multiforme (GBM) has the highest incidence among primary brain tumors, second only to meningioma with a very poor prognosis; and 5-year survival rate of GBM is below 4% from the time of diagnosis [2]. GBM is still one of the most devastating diseases in man and affects more than 17,000 patients in the USA every year [1]. Distant invasiveness is one of the major features of GBM early in its course and makes total neurosurgical resection almost impossible. Therefore, development of novel innovative treatment strategies is urgently needed for GBM. GBM cells depend on glucose and glutamine for energy and growth metabolites, while relying mostly on aerobic fermentation (Warburg effect and the Q-effect); hence, limiting the availability of glucose and glutamine may slow disease progression [3], [4], [5], [6], [7], [8]. KMT (Ketogenic Metabolic Therapy) using calorically restricted ketogenic diets (KD-R), which lower glucose and increase ketone bodies, provide a plausible alternative in blocking malignant cell's energy metabolism because cancer cells cannot effectively burn ketones due to perturbed function and structure of mitochondria [3, 7, 9]. Thus, a metabolic treatment of GBM will target the metabolic ailment shared by all malignant cells (aerobic fermentation), while increasing the vitality of normal brain cells and the entire body [4, [7], [8], [9], [10], [11], [12]]. The KD-R blocks angiogenesis, invasion, inflammation, and stimulates tumor cell apoptosis in mice with GBM [3, 11]. Here, we review evidence suggesting that the saturated fatty acid, caprylic (octanoic) acid (also described as caprylate/octanoate when salt of this fatty acid was used) – an ingredient of ketogenic diets – may be employed at higher doses in treatment of GBM to fortify the efficacy of KMT. The triglyceride precursor of the caprylic acid is tricaprylin/tricaprylate which is also called as trioctanoin/trioctanoate. Figure 1 shows general ketogenesis pathways, Figure 2 shows caprylic acid and its straight-chain isomer valproic acid and Figure 3 shows caprylic acid-modified pathways relevant for cancer treatment.

Caprylic acid is a medium chain (C8) monocarboxylic saturated fatty acid (MCFA) that is enriched in coconut oil and in goat's milk and is also used as an antimicrobial agent in the food industry [13, 14, 15]. Moreover, caprylic acid is a straight-chain isomer of the well-known antiepileptic drug valproic acid [16]. Caprylic acid is a weak acid with a low pH (4.89) that penetrates bacterial cell membranes via passive diffusion and reduces cytosolic pH, likely by stimulating H+-ATPase activity and thereby lowering bacterial cell vitality [14]. Caprylate can be consumed as tricaprylin/tricaprylate (tricaprylate/trioctanoate), a medium chain triglyceride (MCT) [17]. MCTs are partially hydrolysed in the stomach by lingual lipase and by pancreatic lipase in the intestinal lumen with subsequent absorption of MCFAs via the portal vein to the liver [18, 19]. The MCFAs are catabolized mainly to C2 fragments in the liver or utilized to synthesize longer-chain fatty acids [18, 19].

MCFAs enter the mitochondria by a carnitine-independent manner and undergo β-oxidation to provide cellular energy [18, 19]. The MCTs are widely employed in human nutrition for patients with malabsorption syndromes, in infant formulas, and for total parenteral nutrition. The hepatic mitochondrial metabolism of MCFAs such as capric acid yield high levels of acetyl-CoA, which facilitates production of ketone bodies (β-hydroxybutyrate, acetoacetate, and acetone) and CO2, with a minor fraction contributing to the lengthening endogenous fatty acids [18, 20]. Caprylic acid is a part of an endogenous biochemical reaction in which octanoylation occurs by the formation of an ester bond between caprylic acid and a serine residue of the gastric ghrelin hormone [20]. Ghrelin is a 28 amino acid peptide synthesized in the digestive system and and its acylated form binds to the growth hormone secretagogue receptor (GHSR-1a) residing in the pituitary and hypothalamus [20]. Besides regulating gastric acid secretion and gastric motility, caprylated ghrelin controls other physiological pathways including growth hormone secretion, the stimulation of appetite (orexigenic action), glucose homeostasis and adiposity [20]. The increase of caprylated ghrelin levels before meals, although MCFAs do not exist in the stomach lumen, and the shortage of food providing MCFAs, suggest a likely inherent synthesis of the caprylic acid used for ghrelin caprylation [20].

Although the brain contributes only ~2% of adult body weight, it consumes ~22% of the body's oxygen, mainly for aerobic oxidation of glucose [21]. In the healthy and satiated adult, about 97% of the cerebral energy requirement is met by glucose, with the remaining 3% provided mostly by ketones (β-hydroxybutyrate, acetoacetate [AcAc]) [21]. Under the fasted or ketogenic state, fatty acids (especially medium chain fatty acid – MCFA) undergo β-oxidation in the liver and in astrocytes to provide most of the ketones utilized in the brain. MCFAs could traverse the blood–brain barrier; and some authors even claim they can reach brain concentrations that are more than 50% of those of fatty acids in plasma [19, 22]. Although, some authors state that fatty acids are bound to albumin in circulation and can not easily traverse the blood-brain barrier [23, 24]. Under normal states, concentrations of plasma ketones are relatively low (~200 µM), but can increase to during ketogenesis, i.e., prolonged fasting or a very high-fat ketogenic diet, plasma ketones can rise up to 5 mM, at a stage when they provide up to ~ 67% of the cerebral energy requirement [21, 23]. Monocarboxylate transporter-1 in the blood–brain barrier mediates the ketone transfer to the brain [21, 24]. During long-term medically supervised fasting (40–60 d), ketones largely replace glucose, and can supply ~80% of the cerebral energy needs [23, 25, 26].

The high-fat, low carbohydrate ketogenic diet (KD) has been utilized for decades to manage refractory childhood epilepsy and enhances plasma ketones via extreme carbohydrate restriction [26]. Neuronal hyperpolarisation and reduced neuronal excitability can occur during a transition from glucose to ketones as energy source [19]. Another pathway could involve the opening the ATP-sensitive potassium channels thus reducing ATP synthesis from glucose oxidation. In particular, β-hydroxybutyrate may reduce seizures through this cascade in addition to elevating GABA(B) receptor signalling [19]. Other mechanisms may involve the blockage of the mitochondrial permeability transition pore, which causes mitochondrial dysfunction and neuronal apoptosis, and the blockage of adenosine kinase, thereby enhancing levels of adenosine and stimulating the inhibitory A1 receptors of adenosine [19, 27].

Several types of the ketogenic diets exist that can provide about 60–80% of dietary energy with various combinations of fats [19, 28]. The classic KD is very stringent providing very low levels of carbohydrate, and hence, it can be difficult to maintain. Thus, medium-chain triglyceride (MCT) ketogenic diet was designed, in which fats are provided by triglycerides constituting from 60% octanoic acid and 40% decanoic acid (a ten-carbon fatty acid) [19]. In comparison to the classical ketogenic diet, only 45% of dietary energy is supplied by these MCFAs (allowing higher carbohydrate consumption), and the swift metabolism of the shorter fatty acids cause higher production of ketones [19]. According to early suggestions, the anticonvulsant potency of the classical ketogenic diet and of the MCT diet depend upon maintenance of serum levels of acetoacetate above 0.6 mM and of β-hydroxybutyrate above 2 mM [16]. Caprylic acid becomes the most abundant fatty acid in the plasma of epilepsy patients treated with the MCT ketogenic diet and reaches concentrations of 310 to 625 μM (44,64 to 90 µg/ml) [19, 29]. It is important to mention that the glucose ketone index (GKI) calculator was developed as a simple tool to help monitor the efficacy of various ketogenic diets and KMT for managing malignant brain cancer and possibly other cancers that express aerobic fermentation [30].

An MCT diet was administered for four weeks to eight healthy adults (26 ± 1 yrs). The fatty acid content of the MCT diet was 80% caprylic acid, 15% decanoic acid, and 5% lauric acid (12:0)) [18]. Post-MCT, the ketogenic response was proportionally greater for AcAc than for β-hydroxybutyrate, indicating a higher proportion of circulating ketones as AcAc [21]. Unlike β-hydroxybutyrate, AcAc is not dehydrogenated before joining the tricarboxylic acid cycle, so its higher plasma response in comparison to β- hydroxybutyrate may make it a faster energy source [21]. The average sum of AcAc and β-hydroxybutyrate achieved during the metabolic study was anticipated to provide, 8% to 9% of whole cerebral metabolism as ketones [21].

Vandenberghe et al. compared the acute ketogenic effects of various oils in healthy adults: coconut oil [CO; 3% tricaprylin (C8), 5% tricaprin/tridecanoin (C10)], classical MCT oil (55% C8, 35% C10), C8-caprylic acid (95% pure tricaprylin), C10 (95% tricaprin/tridecanoin C10), or CO mixed 50:50 with C8-C10 or C8 [26]. Pure tricaprylin - C8 alone induced the highest plasma ketones for 0–4 and 4–8 h (780 ± 426 μmol . h/L and 1876 ± 772 μmol . h/L, respectively); these values were 813% and 870% higher than controls (P< 0.01) [26]. When pure tricaprylin-C8 was mixed with CO in a 50:50 ratio, the combination lowered the net ketogenic effect by 75% ± 27% in comparison to C8 alone [26]. Hence the 5–10% medium-chain FA content of CO only modestly induced ketone production and only without a meal [26]. In contrast, pure tricaprylin-C8 induced a 3.4-fold higher total plasma ketone response than CO alone [26]. The significant correlation of medium-chain FA intake and plasma ketone response was seen for caprylic acid, but not for decanoic acid, indicating that caprylic acid mediates the ketogenic effect of MCTs containing a mixture of caprylic and decanoic acids [26]. While caprylic acid undergoes β-oxidation in astrocytes more easily than decanoic acid and subsequently more readily causes ketone production, decanoic acid preferentially induces glycolysis, producing lactate [16]. Thevenet et al., examined whether MCFAs exert effects on energy metabolism in pluripotent stem cell–derived human neurons and astrocytes [31]. MCFAs did not lower intracellular ATP levels or induce the energy sensor AMP-activated protein kinase [31]. The MCFA decanoic acid (300 µM) stimulated glycolysis and increased formation of lactate by 49.6%. Caprylic acid (300 µM – 43,2 µg/ml) did not affect glycolysis but increased the rates ketogenesis by 2.17-fold in healthy astrocytes [31]. The authors concluded that the caprylic acid is a better substrate of β-oxidation in astrocytes than is C10 (decanoic acid).

Longer-chain fatty acids can uncouple mitochondria, potentially lowering ATP synthesis and reducing the seizure threshold. MCFAs, on the other hand, are less likely to act as uncouplers [19]. Early studies demonstrated that 400 µM caprylic acid prevented 2,4-dinitrophenol induced mitochondrial uncoupling in hepatocytes [32]. Despite the presence of 2,4-dinitrophenol, caprylic acid enhanced both cytosolic and mitochondrial ATP/ADP ratios, in association with a significant increase in glucose production and decline in lactate+pyruvate flux [32]. It is likely that caprylic acid treatment of uncoupled cells permitted a flux through the malate-aspartate shuttle and the ATP synthase, likely by restoring a mitochondrial proton motive force [32]. Caprylic acid exerts only minimal mitochondrial uncoupling activity even at high concentrations. In contrast to the effects of longer chain faty acids (LCFAs), caprylic acid has minimal effects on the electron transport chain and does not induce lytic damage of biological membranes [33]. Caprylic acid can easily permeate cellular and mitochondria membranes [33]. Caprylic acid also enters the inner mitochondrial membrane and is subsequently activated by matrix-localized acyl-CoA synthetase. It is assumed that caprylic acid generates little reactive oxygen species (ROS) [33]. Caprylic acid or l-octanoylcarnitine (supplemented with only malate or malate+ADP) did not induce H2O2 production from rat brain mitochondria under starvation [33]. Caprylic acid is also a direct precursor of the antioxidant, lipoic acid, an organosulfur compound essential for aerobic metabolism [34]. Lipoic acid is necessary for functioning of several mitochondrial enzyme complexes which include: α-ketoglutarate dehydrogenase (α-KGDH), pyruvate dehydrogenase (PDH), and the glycine cleavage system (GCS) [35].

Oral dosing with caprylic acid enhanced the threshold for myoclonic and clonic convulsions in rats and mice. Caprylic acid significantly increased the seizure threshold in an adenosine-receptor dependent manner under lower blood glucose levels in mice [19]. Benefits of MCFAs may not be limited to epilepsy; for instance, caprylic acid rescues Amyloid-β inhibition of memory consolidation in chicks via providing energetic fuel for astrocytes [36]. Maynard and Gelblum analyzed case records from eight patients with frequent monitoring of cognitive function using the Mini-Mental State Examination (MMSE) and who were treated with tricaprylin (20 gram/day) for ≥6 months [37]. All were outpatients aged ≥50 years, cared for in standard practice, had a diagnosis of AD of mild-to-moderate severity (MMSE 14–24). Response to tricaprylin treatment as defined by MMSE scores varied, yet the rate of decline in MMSE scores appeared slower than published reports for patients treated with classical pharmacotherapy [37]. Ohnuma found similar benefits of caprylic acid triglycerides in patients with mildly effected AD [38]. In parallel, tricaprylin (20 gram/day) increased cognitive functions in ApoE4-negative AD patients with baseline MMSE score ≥14 [38]. These findings suggest that caprylic acid can influence the progression of AD.

Other in vivo studies also give evidence in regard to mitochondria-protecting and neuroprotective efficacies of caprylic acid. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motor neurons which is manifested with progressive muscle weakness and paralysis [13]. ALS patients have perturbed energy balance in association with the reduced enzymes of mitochondrial electron transport chain in spinal cord, indicating that supporting mitochondrial function may be a plausible approach for treatment [13]. When fed tricaprylin, the G93A ALS mouse exerted a significant increase in serum ketones, slower progression of weakness and sparing of spinal cord motor neurons [13]. Parkinson's disease (PD) is a progressive neurodegenerative disorder manifested with the loss of dopaminergic neurons; and damage to the mitochondrial function in the substantia nigra involves in pathogenesis of PD [38]. Caprylic acid significantly hindered the impairment of dopaminergic transmission in the striatum induced by MPTP (1-methyl-4-phenol-1,2,5,6-tetrahydropyridine) toxin and markedly increased PGC-1α expression and PEPCK [38]. PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) is a transcriptional activator that induces the expression of the genes necessary for mitochondrial biogenesis which impairment is associated with the pathogenesis of PD [38]. PGC-1α also activates phosphoenolpyruvate carboxykinase (PEPCK) which increases mitochondrial respiration [38]. Hecker et al., treated starved human endothelial cells and monocytes with caprylate under basal and TNFα-stimulated conditions [39]. TNFα treatment was performed to mimicry the severe inflammatory state in sepsis [39]. Caprylate increased mitochondrial respiratory capacity at baseline and at inflammatory conditions in both cell types, without decreasing mitochondrial DNA content or stimulating the production of proinflammatory cytokine IL-6 [39]. Overall, these results suggest that caprylic acid may differentially effect energetic metabolism in cancerous versus healthy cells with potential double benefits by limiting tumoral fuels and increasing the vitality of neural cells and other benign tissues.

As suggested above, cancer cells depend primarily on glucose and glutamine fermentation to synthesize ATP even under sufficient oxygen, called Warburg effect and the Q-effect, respectively [5, 6, 19]. KMT, using KD-R diets, deprive tumor cells of the glucose needed to drive their glycolytic metabolism [5, 19]. As early as seven decades ago, it was demonstrated that the capacity to oxidize caprylic was lower in mice liver infiltrated with leukemic cells than in healthy liver [40]. When hepatoma tissues were incubated with caprylate-1-14C; major products (comprising more than 5% of the nonvolatile residues) were, in decreasing order, glutamate, di- and tricarboxylic acids, β-hydroxybutyrate, lactate, aspartate, and glutamine [41]. Noteworthy, glutamate-14C to glutamine-14C ratio about 11 fold greater in hepatoma (6.7), than in normal liver (0.58) [41]. This is consistent with the Q-effect and the generation of ATP synthesis through mSLP [5]. Lea and Weber studied the effects of caprylic acid on the glycolytic enzymes of normal liver cells and hepatomas [42]. A %50 inhibition of hexokinase in normal liver occured with higher doses of caprylate (2.9 mM) in comparison to malignant liver hepatoma (1.6 mM) [42]. Glucose reduced caprylic acid activity to block hexokinase in hepatoma cells [42]; in turn, caprylic acid equally blocked lactate production in liver and hepatoma cells treated with glucose [43].

Caprylic acid bypasses the carnitine palmitoyltransferase (CPT) system and enters directly into the mitochondria [17]. The rate of ketone body production from caprylic acid is much lower in rat Fao and H4IIE hepatoma cells than in rat hepatocytes cultured in the same conditions as the majority of the acetyl-CoA synthesized from caprylic acid oxidation is directed into the citric acid cycle, suggested by the high rates of CO2 production [17]. This peculiar phenomenon is also relevant in HepG2 human hepatoma and fetal liver cells [17]; and to our view reflects the feature that cancer cells mimick embryogenesis in regard to many metabolic pathways. Interestingly, caprylic acid levels above 1 mM (144 µg/ml) are considered toxic to embryos; despite their utilization as an energy source in early embryos via mitochondrial β-oxidation [14, 44]. Following incubation with caprylate, the enzyme activities regulating the ketogenic pathway were measured, including acetoacetyl-CoA thiolase, hydroxymethyl-glutaryl-CoA (HMG-CoA) synthase and HMG-CoA lyase in mitochondria from rat hepatocytes and Fao hepatoma cells [17]. The enzyme activity of HMG-CoA synthase was found to be severely depressed (95%) in Fao hepatoma mitochondria in comparison to rat liver mitochondria whereas HMG-CoA lyase activity was similar [17]. In Fao hepatoma mitochondria, the activity of acetoacetyl-CoA thiolase was also depressed by 70% percent [17].

Burton treated B6D2F1/J mice inoculated with intrahepatic implants of M114 cancer cells with intraperitoneal injections of 30 mg sodium caprylate (approximately 1,5 g/kg) [45]. He also treated Nb rats implanted with Nb2 lymphoma with 300 mg tricaprylin (approximately 1,5 g/kg) orally (triglyceride of caprylate, which digestion would yield caprylate) [45]. After 4-11 h of treatment, robust damage to cancer cells occured microscopically whereas healthy hepatic tissue was unaffected. Mice tumors treated once daily from the fourth to eighth day following implantation were obliterated [45]. A caprylic acid preparation applied transdermally to treat subcutaneous implants of hepatoma Nb10L in Nb rats exerted similar damage. These effects were not associated with the tumoral cell proliferation and represented a novel action of anticancer efficacy [45]. In rats treated with tricaprylin, the incidence of mononuclear cell leukemia in the 10 ml/kg tricaprylin group (9/53, 17% incidence) was significantly lesser than that noted for the untreated control group (23/50, 46% incidence) [46]. In Jurkat T cell leukemia and Raji Burkit's lymphoma cells, the maximum tolerated dose of caprylate (in 24hours) is 400 µM; and above levels are toxic [47]. Very importantly, 700 µM (100,8 µg/ml) of caprylic acid was capable to block the growth of human colon, breast and skin cancer cells by about 30%; yet a ten fold higher dose was necessary to achieve similar growth inhibition in benign colon fibroblasts [15].

In human colorectal carcinoma cells, 700 µM of caprylic acid reduced the CDK2 (cyclin-dependent kinase 2) and enhanced the Gadd45 levels, necessary for progression of the cell cycle and execution of apoptosis, respectively [15]. In human colorectal, breast and skin cancer cell lines, 700 µM of caprylic acid reduced cell cycle genes including CKSB1 (CDC 28 protein kinase 1B), CCNA2 (cyclin A2) and CCDN1 (cyclin D), while CDK4 (cyclin-dependent kinase 4) was downregulated only in colon and breast cancer cells [15]. Lastly, the proapoptotic gene P21 (cyclin-dependent kinase inhibitor 1) was also upregulated by 700 µM of caprylic acid in both skin and colon cancer cells. In colorectal carcinoma cells, 700 µM of caprylic acid enhanced the proapoptotic caspase-8 activity [15]. Recent studies also revealed that caprylic acid levels in erythrocyte membrane are lower in skin cancer (basal cell carcinoma) patients than healthy controls [48].

Courage-Maguire et al. performed an analysis on C6 astrocytoma cells to reveal anti-proliferative effects of a series of valproic acid analogs including 2-methylhexanoic acid, 2-ethylhexanoic acid, 4-pentenoic acid, valproic acid, caprylic acid, 2-methyl-2-ethylhexanoic acid, valpromide, 2-N-propyl-4-pentenoic acid, 2-propylhexanoic acid, 2-butylhexanoic acid and 2-ethylbutanoic acid [49]. Very noteworthy, the most potent compounds blocking C6 astrocytoma proliferation were ranked as caprylic acid>2-propylhexanoic acid≥2-ethylhexanoic acid≥valproic acid [49]. Incubation of C6 astrocytoma cells with 2 mM (288 µg/ml) of caprylic acid for 48h was capable to block C6 astrocytoma growth by about 73% percent while the same dose of valproic acid was capable to block C6 astrocytoma growth by about 61% percent [49]. This finding is important as valproic acid was found to be an efficient agent in blocking GBM growth and invasion and there exist epidemiological studies indicating that valproic acid usage in GBM may increase survival [50]. Considering that the C6 glioma cells are much more resistant than human glioblastoma cells against chemotherapeutic and endocrine agents [51, 52], it can be presumed that antitumor effects on human GBM cells may be even more prominent.

High blood glucose directly correlates with worse prognosis in GBM [53], [54], [55], [56] which may associate with profound utilization of glucose in the glycolytic pathway to provide fuel for GBM [10, 12]. Hence, it is important to know whether caprylic acid would influence levels of blood glucose and/or glycolysis. Sanbar et al., investigated the effects of intravenously administered caprylate on glucose turnover in 16 healthy dogs by isotope dilution technics [57]. Intravenous injections of caprylate in concentrations which lead a mean maximal increase of 4.3 mEq/L in plasma FFA levels significantly lowered plasma glucose concentration by an average of 25 mg/100 ml, and lowered the rates of plasma glucose appearance (production) and disappearance (utilization) by 30% and 24% of control values, respectively [57]. Moreover, caprylate infusion reduced glucose significantly by about 34% of controls [57]. The apparent distribution space of glucose was not influenced. The effect on glucose production, mainly the hepatic release of glucose, was higher than the effect on glucose utilization by tissues accounting thereby for the decreases in the intermixing mass [57]. On the other hand, there also exist data suggesting that intraduodenal infusions of caprylate may rise blood glucose [57] or not effect it [58]. A significant decrease in serum glucose concentration was witnessed within 15 minutes after onset of the infusion during continuous injection of sodium caprylate (0.2 M) into weanling and mature rabbits [59]. This relative hypoglycemia persisted for about one hour, after which there was a rebound normoglycemia. Caprylate was inhibitory to the glycolytic enzymes including glucokinase, hexokinase, phosphofructokinase, and pyruvate kinase in rat liver slices [60]. Caprylate also inhibited Glucose-6-phosphate and 6-phosphogluconate dehydrogenases [60]. Moreover, caprylate reduces lactate levels from liver cells of fed rats in association with reduction of hepatic glycolytic pathways [61].

Caprylic acid easily enters the brain (faster than other mono-, di- or tricarboxylic fatty acids) and astrocytes metabolize caprylic acid by β-oxidation [62]. As the glutamine synthetase, which catalyzes the conversion of glutamate to glutamine, localizes predominantly in astrocytes, Kuge et al. analyzed whether a positron-emitting nuclide of caprylate may be employed as a radiopharmaceutical to determine astrocytic functions with positron-emission tomography (PET). Indeed, they demonstrated that in cats, the cerebral tissues were imaged clearly at every level of the brain slices and revealed the ability of 1-11caprylate to serve as a PET tracer for brain imaging [62]. In vitro uptake experiments on human U373-MG GBM cells were employed to reveal the potential use of [1-11C]caprylate as a PET-tracer [63]. Uptake of [1-14C]caprylate enhanced until 60 min after application in a time dependent manner. The uptake of [1-11C]caprylate demonstrated similar findings to that of [1-14C]caprylate until 10 min; [1-14C]caprylate uptake enhanced gradually with the reduction in pH [63]. Glucose deprivation, hypoxia, or gluatamate did not effect the [1-14C]caprylate uptake [63]. The enhanced uptake of caprylate with enhanced acidity is of potential interest since the core of tumors are highly acidic due to higher anaerobic metabolism and production of lactate.

The potential utility of employing [1-11C]caprylate as a PET tracer for imaging and assessing the extent of ischemic stroke was also investigated [64]. Dogs were implanted with a catheter in the left internal carotid artery (LICA) and an autologous blood clot was injected into the LICA. Using a PET scanner, the brain distribution of [1-11C]caprylate and cerebral blood flow (CBF) were determined 24h after the insult [64]. Post mortem cerebral tissues unstained with 2,3,5-triphenyltetrazolium chloride were determined as infarcts. In the infarct regions, uptake of [1-11C]caprylate reduced simultaneously with the reduction of CBF [64]. In opposite, normal accumulation of [1-11C]caprylate was witnessed in ischemic but vital areas, indicating that an enhanced uptake of [1-11C]caprylate relative to CBF occurs in these regions [64]. Cerebral distribution of [1-11C]caprylate was also determined and compared with CBF by employing rat and canine models of middle cerebral artery (MCA) occlusion and PET [65]. In rats, the cerebral distribution of [15O]H20 determined 1-2 h and 5-6 h after occlusion was compared with that of [1-11C]caprylate determined 3-4 h after occlusion. Radioactivity ratios of ischemic to normal hemispheres revealed with [15] H2O were less than those demonstrated with [1-11C]caprylate which were confirmed by the canine experiments of MCA-occlusion [65]. 24-hours after insult, CBF declined in the occluded MCA-territory, whereas normal or higher uptake of [1-11C]caprylate was witnessed in the ischemic areas. Hence, it is suggested that [1-11C]caprylate-uptake measurements may provide additional functional information different from CBF [65]. The authors attributed the enhanced uptake of [1-11C]caprylate to the responses against ischemia, acidosis development in the hypoxic areas and to the changed glutamate turnover [65]. The [1-11C]caprylate accumulation in ischemic, but vital regions, reveals its potential for imaging and assessing ischemic brain areas. This feature may also be helpful in imaging hypoxic areas within the brain tumors. Experiments on rat brain slices once again demonstrated that the selective inhibitor of glial metabolism, fluoroacetate, reduced the uptake of [1-14C]caprylate suggesting that the major cells which uptake (and probably metabolize) caprylic acid are astrocytes [66]; suggesting its important potential to reveal metabolism of astrocytes either of benign and malignant origin.

Section snippets

Oral Toxicity

No deaths were found in mice treated with up to 25 ml/kg of Miglyol 812 (caproic acid maximum 3%, caprylic acid 50-65%, capric acid 30-45%, lauric acid maximum 5%) [18]. On the other hand, three deaths were seen in mice treated with 20-25 ml/kg Miglyol 810 (slightly higher content of C8 fatty acids than Miglyol 812) [18]. Nonetheless, all other symptoms vanished in the survivors at the day 3. No toxicity was observed following 10-days treatment with 4.5-36 ml/kg Miglyol 812 in fasted Wistar

Declaration of Competing Interest

None to declare.

Acknowledgements

The author would like to acknowledge support from the Foundation for Metabolic Cancer Therapies, the Claudia & Nelson Pleltz Foundation, Crossfit Inc., Lewis Topper, Edward Miller, Dr. Joseph Maroon and the Boston College research expense fund.

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