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TRANSLATIONAL PHYSIOLOGY
Departments of 1Pediatrics and 2Nutrition, Case Western Reserve University, Cleveland, Ohio; 3Fachhochschule Lippe und Hoxter University of Applied Sciences, Detmold, Germany; 4Sasol GmbH, Witten, Germany; and 5Institute of Metabolic Disease, Baylor University Medical Center, Dallas, Texas
Submitted 5 August 2005 ; accepted in final form 8 May 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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heptanoate; C5-ketone bodies; fatty acid oxidation disorders; anaplerosis
-oxidation spiral. The most common disorders of -oxidation affect very-long-chain acyl-CoA dehydrogenase, mitochondrial trifunctional protein, isolated long-chain hydroxyacyl-CoA dehydrogenase, and medium- and short-chain acyl-CoA dehydrogenase. Patients with long-chain FOD commonly present with recurrent hypoketotic hypoglycemia, hypertrophic or dilated cardiomyopathy, cardiac arrhythmias, rhabdomyolysis, muscle weakness, and hypotonia, (for a review, see Ref. 21). There is considerable phenotypic variation associated with nearly all of these disorders.
The classical chronic treatment of long-chain FOD involves frequent feeding with a diet adjusted so as to lower long-chain fat intake from the usual 30–35% of total kilocalories to 
20%, including essential fatty acids. The decrease in energy from long-chain fats is partly compensated by an increase in carbohydrates, often with cornstarch at bed time. In addition, even carbon medium-chain triglycerides (trioctanoin/tridecanoin) are added to the diet. This is because fatty acids with 8–10 carbons enter the mitochondrion as carboxylates, which, after activation, require only those -oxidative enzymes with medium- and short-chain length specificity. The dietary treatment with medium-chain triglycerides is obviously restricted to long-chain disorders and is contraindicated for medium- and short-chain deficiencies.
A new strategy was recently conceived for the dietary treatment of long-chain FOD, i.e., providing about one-third of the calories as triheptanoin (22). The catabolism of heptanoate yields anaplerotic propionyl-CoA in addition to acetyl-CoA. It was hypothesized that part of the energy deficit in FOD patients results from a decrease in the concentration of citric acid cycle intermediates in muscle and heart cells. These intermediates carry the carbons of acetyl groups as they are oxidized. The treatment with triheptanoin resulted in a rapid improvement of muscle and cardiac function in the three long-chain FOD patients described in Ref. 22, as well as in other patients who are presently involved in clinical trials (Roe CR, unpublished observations). Also, treatment with triheptanoin improved for 6 mo the condition of a patient with almost complete deficiency of liver pyruvate carboxylase (a major contributor to hepatic anaplerosis) before she succumbed to an acute episode of gastroenteritis (17).
In the liver, propionyl-CoA derived from heptanoate oxidation has two main fates. First, it is gluconeogenic via succinyl-CoA and part of the citric acid cycle reactions. Second, it is converted to the C5-ketone bodies -ketopentanoate (BKP) and -hydroxypentanoate (BHP) via the hydroxylmethylglutaryl (HMG)-CoA cycle. The C5-ketone bodies are analogs of the C4 acetoacetate (AcAc) and -hydroxybutyrate (BHB). In peripheral tissues, the C5-ketone bodies are converted to acetyl-CoA and anaplerotic propionyl-CoA by the combined action of D--hydroxybutyrate dehydrogenase, 3-oxoacid-CoA transferase, and acetoacetyl-CoA thiolase. Thus, after administration of heptanoate, propionyl-CoA and acetyl-CoA are formed in peripheral tissues by 1) -oxidation of heptanoate and 2) utilization of C5-ketone bodies formed in the liver.
The goal of the present study was to test in normal rats the metabolism of a triheptanoin preparation that might be used in the future for the treatment of acute long-chain FOD decompensation, via either intravenous or enteral infusion. An emulsion of triheptanoin was infused intravenously (iv) or intraduodenally (id), the latter to delineate the role of first-pass metabolism of the substrate through the portal drainage system. Our data show that the route of administration has a major impact on the distribution of triheptanoin metabolites in systemic blood.
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MATERIALS AND METHODS |
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[U-13C6]glucose, [6,6-2H2]glucose, [2H8]glycerol, [U-13C3]glycerol, [
-2H3]heptanoic acid, 2H2O, NaO2H, sodium borodeuteride (NaB2H4), and general chemicals were purchased from Isotec/Sigma-Aldrich. Pentafluorobenzyl bromide was purchased from Pierce. An internal standard of R,S--hydroxy-[2H5]pentanoate was prepared by incubating ethyl -ketopentanoate in 2H2O plus NaO2H, followed by reducing -keto-[2H4]pentanoate with sodium borodeuteride (16). A 10% sterile emulsion of triheptanoin was kindly donated by Sasol Germany. As per Sasol's quality control data, the heptanoic acid used to synthesize triheptanoin contained 99.5% heptanoic acid, with traces of hexanoic, octanoic, and decanoic acids. The triheptanoin used to prepare the emulsion contained 97.5% triester, with the remaining 2.5% being monoester and diester. The emulsion was stabilized with 1% Imwitor 2020 (Sasol), a food-approved emulsifier prepared by interesterification of a mixture of compounds with the following weight composition: 14% glycerol, 18.5% tartaric acid, 15% acetic acid, 4.5% palmitic acid, 2.2% stearic acid, 14.5% oleic acid, 28.2% linoleic acid, and 3.1% linolenic acid. The emulsifier meets the specifications of the Food Chemical Codex (3rd ed.) (3a).
Animal Experiments
The animal experiments were approved by the Institutional Animal Care and Utilization Committee of the Case School of Medicine.
Preparation of rats. Male Sprague-Dawley rats were fed ad libitum with Harlan-Teklad rat chow. After an overnight fast, rats (235–317 g) were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg dissolved in water). Catheters were inserted in a jugular vein and the ventral tail artery. Some rats were also fitted with a catheter in the duodenum (through a small laparotomy). After successful placement of catheters, saline (3.5 ml/h) and a maintenance dose of pentobarbital (5.8 mg/h) were infused into the jugular vein until the end of the experiments. A basal blood sample (70 µl) was obtained from the tail artery before the start of each protocol.
Protocol 1 (6 groups of 6–7 rats). Saline or a 10% triheptanoin emulsion was infused for 90 min either via the jugular vein at 0, 10, 20, 30, or 40% of caloric requirement or intraduodenally at 40% of caloric requirement. Arterial blood samples (70 µl) were taken at 60, 70, 80, and 90 min. At 91 min, 0.5 ml of blood was sampled from the abdominal aorta, after which the animal was killed by exsanguination.
Protocol 2 (4 groups of 7–9 rats). Rats were prepared as in protocol 1. They were infused for 120 min either with intravenous saline (group 1), intravenous triheptanoin (group 2, 40% of caloric requirement), or intraduodenal triheptanoin (groups 3 and 4, 40% of caloric requirement). In addition, they were infused intravenously with [6,6-2H2]glucose (1.2 µmol·min–1·kg–1) and with [U-13C3]glycerol (1.5 µmol·min–1·kg–1 in group 1, and 3.7 µmol·min–1·kg–1 in the triheptanoin groups 2, 3, and 4). In group 4, the tracers were infused intraduodenally with the triheptanoin emulsion. Arterial blood samples (70 µl) were taken at 0 (basal), 90, 100, 110, and 120 min. At 121 min, 0.5 ml of blood was again sampled from the abdominal aorta and the animals were killed as above.
Analytic procedures.
All arterial blood samples (70 µl) were immediately pipetted into glass tubes containing a 1-ml aqueous solution of internal standards: [-2H3]heptanoate (30 nmol), R,S--hydroxy-[2H6]butyrate (34 nmol), R,S--hydroxy-[2H5]pentanoate (28 nmol), and (for protocol 2) [U-13C6]glucose (50 nmol) and [2H5]glycerol (2 or 5 nmol). After quick mixing, the solution ofhemolyzed blood was treated with 40 µl of 1 M sodium borodeuteride in 0.1 M NaOH. The treatment with sodium borodeuteride converts unstable AcAc and BKP to the stable M1 BHB and M1 BHP, respectively, which can be distinguished by GC-MS from the unlabeled BHB and BHP present in blood (16). It also converts glucose to the M1 sorbitol. After 30 min, protein denaturation was achieved with 50 µl of saturated sulfosalicylic acid. In those experiments measuring glucose and glycerol, samples were divided prior to the addition of sulfosalicylic acid and HCl by transferring 0.2 ml of solution to a separate tube. The glucose and glycerol tubes were then acidified with HCl, whereas sulfosalicyclic acid was added to the remaining tubes. Samples were stored at –20°C until assayed.
Assay of heptanoate and C4- and C5-ketone bodies.
After treatment with sulfosalicylic acid, the slurry was acidified with HCl and extracted with 3 x 4 ml of diethyl ether. The combined extract was dried over Na2SO4, evaporated down to 50 µl, and sonicated for 1 h with 3.5 µl of pentafluorobenzyl bromide, 10 µl of pyridine, and 37.5 µl of acetonitrile. After extraction with 3 ml of hexane, drying the extract over Na2SO4, and evaporating the solvent, the residue was reacted with 15 µl of of acetic anhydride and 30 µl of pyridine in 285 µl of ethyl acetate at room temperature overnight. This procedure converts 1) fatty acids to pentafluorobenzyl derivatives and 2) hydroxyacids to acetyl-pentafluorobenzyl derivatives. After extracting with diethyl ether, drying over Na2SO4 and evaporating with solvent, the residue was dissolved in 40 µl of ethyl acetate and 2 µl injected into an Agilent gas chromatograph linked to a 5973 MSD mass spectrometer. The chromatograph was equipped with a 30-m 77-OV-225 capillary column (Quadrex). The carrier gas was helium (30 ml/min), and the injection mode was splitless. The GC injector temperature was set at 190°C, and the transfer line was held at 280°C. The column temperature was increased from 100°C by 5°C/min to 235°C and held for 20 min. The mass spectrometer was operated under ammonia negative chemical ionization with the source pressure adjusted to obtain the maximal signal. The retention times and ions monitored were as follows: heptanoate (16 min, m/z 129, 132); BHB (20 min, m/z 145, 146, 151); BHP (21 min, m/z 159, 160, 164).
Assay of glucose and glycerol. After the addition of 1 drop of 12 N HCl, the solution was evaporated under nitrogen. The residue was reacted with 150 µl of acetic anhydride and 300 µl of pyridine, heated for 5 min at 100°C, and then left overnight at room temperature. The next day, samples were extracted with ethyl ether, and the extract was dried over Na2SO4 before evaporation. The residue was dissolved in 40 µl of ethyl acetate and either 2 µl (glucose assay) or 0.2 µl (glycerol assay) injected into the same chromatograph as above. The injection mode was either splitless (glucose) or split (glycerol). The column temperature was increased from 100°C by 20°C/min to 190°C, by 5°C/min from 190 to 220°C, and then by 20°C/min from 220 to 235°C, where it was held for 45 min. The mass spectrometer was operated under ammonia positive chemical ionization with the source pressure adjusted to obtain the maximal signal. The retention times and ions monitored were as follows: glycerol (8 min, m/z 236, 237, 238, 239, 241), glucose converted to sorbitol (31 min, m/z 453, 454, 455, 456, 459).
Data Analysis and Calculations
Total C4-ketone bodies were calculated as the sum of BHB plus AcAc, whereas total C5-ketone bodies were the sum of BHP plus BKP. Glucose and glycerol rates of appearance (Ra) were calculated (15) according to the steady-state equation:
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In the first study (protocol 1), we infused the triheptanoin emulsion for 90 min at 1) 0–40% of the caloric requirement intravenously or 2) 40% of the caloric requirement intraduodenally. Figure 1 shows that the intravenous infusion of triheptanoin (at 10–40% of caloric requirement) resulted in fairly stable blood heptanoate concentrations ranging from 0.07 to 0.5 mM. In contrast, the intraduodenal infusion of triheptanoin at 40% of the caloric requirement resulted in a very low blood heptanoate concentration, i.e., 0.02 mM. The blood heptanoate concentrations were undetectable (P < 0.005 mM) in basal samples and in samples from saline-infused rats. Because of the small sample of blood that we were able to obtain from each rat, blood concentrations of triheptanoin were not obtained.
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0.22 mM; Fig. 2, top curve).
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0.1 mM in conscious overnight-fasted rats to 1 mM (25). The high basal concentration of C4-ketone bodies continued to increase significantly in saline-infused rats (P < 0.05), again presumably as a result of the prolonged fasting and anesthesia, but decreased significantly during the intravenous infusion of triheptanoin at 30 and 40% of the caloric requirement (P < 0.001).
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The blood glucose concentration (Fig. 4A) was not significantly affected by the intravenous or intraduodenal infusion of triheptanoin except at 100 min, when glucose concentration was higher with intraduodenal triheptanoin infusion than with intravenous infusion (P < 0.05). However, the rate of glucose appearance (glucose Ra; Fig. 4B) was significantly increased by about one-half by the intraduodenal infusion of triheptanoin (P < 0.05).
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0.15 mM (20)]. This most likely resulted from the stress of the anesthesia and surgery, which stimulates lipolysis (5, 6). These effects are presumably mediated via increased concentrations of catecholamines and cortisol. Table 1 shows the detail of the calculations of the Ra of glycerol in the four groups of rats of protocol 2. In saline-infused control rats, the glycerol Ra (21.7 µmol·min–1·kg–1) was similar to data from the literature (1). The intravenous infusion of triheptanoin tripled the arterial blood glycerol concentration (Fig. 4C) and about doubled the endogenous glycerol Ra (group 2; Table 1) compared with the saline control. In contrast, the intraduodenal infusion of triheptanoin did not affect the arterial blood glycerol concentration (Fig. 4C). This suggests that, during the intraduodenal infusion of triheptanoin, the glycerol derived from the intestinal hydrolysis of triheptanoin was cleared by the liver. Therefore, to calculate the endogenous glycerol Ra, one should not deduct from the total glycerol Ra (endogenous + exogenous, 25.4 µmol·min–1·kg–1) the amount of glycerol infused intraduodenally as part of the triheptanoin emulsion. So, the endogenous Ra of glycerol of 25.4 µmol·min–1·kg–1 is a reasonable first approximation of peripheral lipolysis during the intraduodenal infusion of triheptanoin. A small correction to this Ra will be added below.
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First, in experiments with intraduodenal infusion of triheptanoin and intravenous infusion of tracer glycerol (group 3; Table 1), from the total glycerol Ra (25.4 µmol·min–1·kg–1, i.e., endogenous + exogenous), one deducts 16% of the rate of glycerol administration via triheptanoin:
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Figures 1 and 2 show that, during the intraduodenal infusion of triheptanoin, virtually no heptanoate derived from triheptanoin hydrolysis escaped uptake by the liver. This is consistent with reports of essentially complete hepatic clearance of enterally administered medium-chain triglycerides (2, 9). It appears that a large fraction of the heptanoate reaching the liver is oxidized to BKP-CoA, which is partly converted to the C5-ketone body BKP in the HMG-CoA cycle. BKP then equilibrates with BHP via a reaction catalyzed by BHB dehydrogenase. Indeed, standard solutions of unlabeled and labeled BHP and BKP are assayed with BHB dehydrogenase (16).
The total concentration of C4- and C5-ketone bodies (Figs. 2 and 3) did not exceed 1.2 mM in any group. Thus the intravenous infusion of triheptanoin at up to 40% of the caloric requirement did not induce a toxic ketosis.
The decrease in the concentration of C4-ketone bodies during the intravenous infusion of triheptanoin at 30 or 40% of the caloric requirement was quite puzzling, especially in view of the reported increase in C4-ketone bodies during infusion of even-chain triglycerides (medium- or long-chain) (3, 19, 23, 24). Because the heptanoate molecule has two acetyl moieties, these were expected to contribute somewhat to the production of C4-ketone bodies. We considered the possibility that the intravenous infusion of triheptanoin could result in an inhibition of peripheral lipolysis, with a subsequent decrease in C4-ketogenesis. We thus decided to assess the effect of triheptanoin on lipolysis in another series of experiments by measuring the Ra of glucose and glycerol. The glucose Ra was significantly increased by about one-half by the intraduodenal infusion of triheptanoin (P < 0.05; Fig. 4B). The increase in glucose Ra (10 µmol·min–1·kg–1) was only about one-third of the potential glucose equivalent of the triheptanoin infused (glycerol + propionyl-CoA moieties). This is because part of the propionyl-CoA moiety of heptanoate was converted to C5-ketone bodies (Fig. 2). During the intravenous infusion of triheptanoin, the glucose Ra was not significantly increased (Fig. 4B), because a large fraction of the heptanoate derived from triheptanoin hydrolysis in blood was presumably taken up by nongluconeogenic peripheral tissues. In contrast, during the intraduodenal infusion, all the glycerol and heptanoate derived from triheptanoin hydrolysis in the gut reached the liver, the main gluconeogenic organ.
The activation of lipolysis during the intravenous infusion of triheptanoin might result from the activation of lipoprotein lipase by the infused triglyceride. Karpe et al. (14) showed that intravenous infusion of long-chain triglycerides in humans leads to an up to ninefold activation of lipoprotein lipase in arterialized blood. Nordenstrom et al. (18) demonstrated a similar increase when a combination medium-chain/long-chain triglyceride infusion was infused, with greater lipoprotein lipase activities noted with the medium-chain/long-chain infusion than with long-chain infusion alone. In this latter set of experiments, there was a significant correlation (r = 0.77) between plasma triglyceride concentrations and lipoprotein lipase activity during triglyceride infusion.
In our experiments where triheptanoin was infused intraduodenally (groups 3 and 4; Table 1), there was no increase in endogenous glycerol Ra, presumably because triglyceride particles did not enter the vascular bed and thus did not activate lipoprotein lipase.
During the intravenous infusion of triheptanoin, the activation of lipolysis presumably led to the release of long-chain fatty acids from adipose tissue. However, the arterial concentration of C4-ketone bodies decreased. This is an apparent contradiction, as an increase in the supply of long-chain fatty acids to the liver would be expected to result in enhanced C4-ketogenesis. We suggest that the bulk of the long-chain fatty acids released by lipolysis was reesterified, whereas heptanoate was preferentially oxidized. Preferential oxidation of heptanoate would be favored because 1) it does not accumulate in adipose tissue (26) and 2) its oxidation bypasses the carnitine palmitoyltransferase system. Reesterification of long-chain fatty acids occurs partly in the liver, where the glycerol moiety of triheptanoin is activated to 
-glycerophosphate, an intermediate of triglyceride synthesis. However, there are other sites of reesterification (13), in particular, muscle, which uses plasma glycerol for triglyceride synthesis (10) and which is a site of triglyceride storage (8, 12). Reesterification of long-chain fatty acids in muscle was probably promoted by 1) the tripling in glycerol concentration during the intravenous infusion of triheptanoin (Fig. 4C) and 2) the supply of energy substrates in the form of heptanoate and C5-ketone bodies. The increase in glucose Ra and the slight hyperglycemia induced by triheptanoin infusion may have led to the release of insulin, which promoted reesterification of long-chain fatty acids in muscle and adipose tissue. Because fatty acids shorter than nine carbons are not stored as glycerides in the rat (26), the heptanoate moiety of triheptanoin was presumably oxidized in preference to the long-chain fatty acids released by enhanced lipolysis. Experiments in dogs fitted with duodenal, transhepatic, and transmuscle catheters are planned to test the above-mentioned mechanisms.
The data of this translational physiology study have a number of clinical implications for the potential use of triheptanoin emulsion in the treatment of long-chain FOD decompensation. Currently, only the enteral form of triheptanoin is available for therapy, as part of semisolid meals or baby formula. However, parenteral triheptanoin has the potential to be used during an acute decompensation or during episodes of malabsorption. The production of propionyl-CoA from heptanoate and/or C5-ketone bodies would replenish the pool of citric acid cycle intermediates and restore energy production. Because the heart is the organ with the highest activity of 3-oxoacid-CoA transferase (7), it forms propionyl-CoA from both heptanoate and C5-ketone bodies.
The data of Figs. 1 and 2 show that the intravenous and intraduodenal infusions of triheptanoin lead to opposite blood concentration ratios of heptanoate to C5-ketone bodies. In some clinical conditions, one might prefer to administer triheptanoin intraduodenally to favor supplying C5-ketone bodies over heptanoate to peripheral tissues. It is likely that C5-ketone bodies, like C4-ketone bodies, can be taken up and used by the brain,1 which has very high activities of 3-oxoacid-CoA transferase and BHB dehydrogenase (7). The intravenous administration of triheptanoin leads to an activation of lipolysis, which would not be desirable during the acute treatment of long-chain FOD decompensation. However, this stimulation of lipolysis could then be countered by the reesterification of long-chain fatty acids. Reesterification would be enhanced by the infusions of glucose and insulin that are used in severe cases of FOD decompensation, so the addition of parenteral triheptanoin to the current treatment by glucose and insulin may be beneficial to these patients. In mild cases of FOD decompensation, enteral administration of triheptanoin emulsion would be preferred, in the absence of diarrhea.
We want to stress that we do not imply that this study, conducted in anesthetized rats, clears the way for the utilization of triheptanoin emulsion for the treatment of decompensated patients with long-chain FOD. This will require additional studies in larger animals with metabolic rates per kilogram closer to those of humans. Although heptanoate does not accumulate in tissue glycerides (26), detailed studies on the influence of triheptanoin infusion on the profile and kinetics of plasma lipids, including the concentration of plasma triheptanoin, will need to be conducted in various species, including in genetically engineered mice with FOD.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 The improvement of the neurological status of a child with pyruvate carboxylase deficiency during treatment with triheptanoin (17) suggests that C5-ketone bodies are taken up and used by the brain. 
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REFERENCES |
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-hydroxypentanoate and of -ketopentanoate in conscious dogs. Am J Physiol Endocrinol Metab 268: E446–E452, 1995.
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