Treatment of cardiomyopathy and rhabdomyolysis in long-chain fat oxidation disorders using an anaplerotic odd-chain triglyceride (original) (raw)

Current treatment of long-chain fat oxidation disorders. The plasma membrane carnitine transporter defect is the only one of the mitochondrial fat oxidation disorders for which an effective treatment (carnitine supplementation) is essentially a cure, since mitochondrial β-oxidation is intact. Current treatment for the other long-chain fat oxidation disorders, including VLCAD deficiency, is designed to (a) reduce the amount of long-chain dietary fat while covering the needs for essential fatty acids, (b) provide carnitine to convert potentially toxic long-chain acyl-CoAs to acylcarnitines, (c) provide a large fraction of the calories as carbohydrates (including cornstarch at night) to reduce body fat utilization and prevent hypoglycemia, and (d) provide about one-third of the calories as even-chain MCT. The recommended use of even-carbon MCT for treatment of these disorders is based on the notion that the metabolism of the eight to ten carbon fatty acids in MCT oil is independent of CPT I, translocase, CPT II, VLCAD, trifunctional Protein, and LCHAD enzyme activities. It has been assumed that the even-carbon medium-chain fatty acids simply enter the mitochondrion as carboxylates before activation and, therefore, require only those β-oxidative enzymes with shorter chain-length specificity for further oxidation and energy production. A significant advantage of MCT dietary therapy for long-chain fat oxidation disorders is their conversion to C4 ketone bodies by the liver for export as an energy source for other organ systems, including the brain. However, although MCT oil is a good source of calories for patients with long-chain fat oxidation disorders, it does not meet the needs of those patients prone to recurrent hypoglycemia. The even-carbon fatty acids in MCT (octanoate and decanoate), like all even-carbon fatty acids, are not gluconeogenic. Although the glycerol moiety of MCT oil is gluconeogenic, the amount of glucose it can generate is very small.

It is well recognized that fasting, often due to infection, results in profound hypoglycemia and massive lipid mobilization that lead to severe symptoms such as cardiomyopathy, rhabdomyolysis, and sudden death (14). Avoidance of fasting, frequent feeding (increased carbohydrate), and supplemental bedtime cornstarch (originally introduced for therapy of type I glycogenosis) (15) are all recommended to prevent lipolysis. The use of MCT in the diet has been reported to alleviate acute cardiomyopathy with pericardial effusion in acutely ill VLCAD patients with significant, but not always acceptable, recovery, as seen in our patient (16, 17). Currently, during an acute infection or illness secondary to fasting, the only recourse is intravenous glucose, often with insulin infusion, to control the associated stimulation of lipolysis.

Patient studies. Before any clinical intervention, fibroblasts from the three patients were cultured with [ω-2H3]fatty acids (even- and odd-chain) to determine which, if any, fatty acids might be used in the diet (Table 1). Incubations with [ω-2H3]pentadecanoate or [ω-2H3]palmitate resulted in similar accumulations of pentadecanoyl- and palmitoyl-carnitine. This indicated that pentadecanoyl-CoA, like palmitoyl-CoA, is a substrate for the defective VLCAD enzyme. Incubation of [ω-2H3]odd-carbon substrates with fibroblasts always results in a much greater accumulation of [ω-2H3]propionylcarnitine than is seen in blood acylcarnitine profiles (<2.64 μM). The slow utilization of propionyl-CoA reflected by the accumulation of C3 is not understood. However, it allows comparison of the relative production of propionyl-CoA, in vitro, from different odd-carbon substrates. One possibility is that the concentrations of other anaplerotic substrates (glucose, glutamate, etc.) are so high in the culture medium that entry of propionyl groups into the CAC is impaired.

In normal cells, [ω-2H3]pentadecanoate (C15) is associated with the greatest amount of [ω-2H3]propionylcarnitine. However, in VLCAD-deficient cells, the amount of [ω-2H3]pentadecanoyl-carnitine that accumulates suggests that it is not suitable as a potential dietary substrate. The amount of [ω-2H3]propionylcarnitine produced from [ω-2H3]heptanoate is also high and much greater than that produced from [ω-2H3]nonanoate (C9). This relationship is extremely reproducible. The VLCAD-deficient cells produced much less [ω-2H3]propionylcarnitine than did normal cells when incubated with [ω-2H3]pentadecanoate. In the absence of the VLCAD enzyme in the mitochondria, it is possible that only the smaller peroxisomal contribution to the oxidation of [ω-2H3]pentadecanoate can be observed (18). The results with [ω-2H3]heptanoate (C7) suggested that heptanoate (as a triglyceride) was the obvious candidate for dietary therapy because (a) normal amounts of [ω-2H3]propionylcarnitine were produced by the VLCAD-deficient cells and (b) there was no significant accumulation of [ω-2H3]heptanoyl-carnitine.

The results of the chain-length panel incubations clearly demonstrated that [ω-2H3]heptanoate was converted to [ω-2H3]pentanoyl-CoA and then to [ω-2H3]propionyl-CoA in this VLCAD cell line. The activation of heptanoate is probably catalyzed by the medium-chain acyl-CoA synthetase. The initial oxidation of heptanoyl-CoA is most likely catalyzed by medium-chain acyl-CoA dehydrogenase. [ω-2H3]pentanoyl-CoA is oxidized to 3-keto-[ω-2H3]pentanoyl-CoA, which is cleaved to acetyl-CoA and [ω-2H3]propionyl-CoA by the short-chain 3-ketoacyl-CoA thiolase (acetoacetyl-CoA thiolase). This is evidenced by the production and accumulation of [ω-2H3]propionylcarnitine in vitro.

Further evidence for the above was provided by the results of in vivo testing of patient 1 (as well as patients 2 and 3) with meals containing MCT oil or triheptanoin (Tables 2 and 3). During the meal with MCT oil, the plasma and urine metabolite profiles were consistent with previous experiences in that there was a rapid appearance of the four-carbon ketone bodies (BHB and AcAc). The five-carbon ketone bodies (BHP and BKP) were not detected in plasma, and there was no increment in C3. As expected, both octanoate and suberate were excreted in the urine. During the meal with triheptanoin, there was a rapid appearance of both the four-carbon and the five-carbon ketone bodies. The latter reached peak levels in 90–120 minutes with a simultaneous peak of C3. There were no significant increases in heptanoyl- or pentanoyl-carnitine. Plasma heptanoate achieved a peak level similar to that of plasma octanoate with the MCT oil meal. The urine studies reflected the expected increased excretion of BHB, AcAc, BHP, heptanoate, and pimelate, each derived from triheptanoin. These results confirm the integrity of the proposed pathway described from patient 1’s fibroblasts.

There was no change in these patients’ clinical status during the days they received MCT oil. However, within 5–12 hours of the first meal containing triheptanoin, there was an almost immediate improvement in muscle strength, endurance, and activity. The increased performance, resolution of hepatomegaly, and cardiomyopathy in patient 1 has persisted during the 26 months of her trial without any major episodes of rhabdomyolysis. A similar improvement (15 months without any hospitalizations) was experienced by patient 2 until the reintroduction of steroid-containing medications. Patient 3 also experienced significant improvement in muscle strength and endurance during the 4-month trial of dietary triheptanoin. The return to conventional therapy was associated with renewed episodes of rhabdomyolysis. These dramatic and persistent improvements support the notion that the primary problem in VLCAD deficiency and other long-chain fat oxidation disorders is the severe energy deficit leading to chronic and acute cardiomyopathy (19) and recurrent rhabdomyolysis.

There has been no evidence for any chemical toxicity from the triheptanoin diet during the 26-month treatment interval involving these three patients. Serial studies of blood chemistries (including acylcarnitines) and urinary organic acid analysis have shown no significant abnormalities. The theoretical early concerns of possible propionate overload and “iatrogenic propionic acidemia” were soon eliminated, as illustrated in Figure 2. The levels of 3-hydroxypropionate, propionylglycine, and methylcitrate were orders of magnitude lower than those observed with serial samples from patients with propionic acidemia. Surprisingly, propionylglycine was not detected in any of the samples from the patients. This suggests that the propionyl-CoA pool does not increase sufficiently in hepatocytes oxidizing heptanoate to form this glycine conjugate. (The glycine-_N_-acylase was intact in the patients as evidenced by hippurate [benzoylglycine] excretion.) It also suggests that much of the oxidized heptanoate is converted to the five-carbon ketone bodies (BHP and BKP) in the liver.

Each of these patients experienced rapid weight gain, initially. Since patients with VLCAD deficiency are frequently placed on a low-fat high-carbohydrate diet, the excess carbohydrate while receiving triheptanoin appears to be responsible for the weight gain. When the carbohydrate content is reduced to more normal levels, the weight gain is actually curtailed and actual weight loss can occur in the absence of any symptoms (patients 1 and 2). There has been no weight problem with subsequent patients in this dietary protocol. The triheptanoin diet is initiated with normal, not augmented, amounts of carbohydrate.

Why is triheptanoin more effective than trioctanoin for treatment of long-chain fat oxidation disorders? The remarkable improvement in the clinical and biochemical condition of patient 1 with VLCAD deficiency occurred almost immediately, when her dietary treatment with MCT oil was changed to treatment with triheptanoin. Given the similarity of structure of octanoate and heptanoate, one has to consider differences in their metabolism and metabolic effects.

The metabolism of octanoate has been investigated much more thoroughly than that of heptanoate. This is because very small amounts of odd-chain fatty acids are present in most human diets. However, there is an obvious difference in the metabolism of the two compounds: the catabolism of octanoate leads only to acetyl-CoA, while the catabolism of heptanoate leads to acetyl-CoA and propionyl-CoA. The provision of propionyl-CoA has two important metabolic consequences: (a) propionyl-CoA is an anaplerotic substrate for the CAC in all tissues, and (b) it is gluconeogenic in liver and kidney cortex. Increasing the supply of propionyl-CoA or of other anaplerotic precursors may have little impact on the biochemical and physiological homeostasis, as well as on the maximum exercise capacity, of a healthy subject (20, 21). However, it could be crucial in patients with VLCAD deficiency and other long-chain fat oxidation disorders. Indeed, patients with these defects have a clear decrease in their ability to generate energy in their muscle and/or heart (chronic muscle weakness, cardiomyopathy, rhabdomyolysis). This occurs despite a high-carbohydrate, low-fat diet supplemented with MCT oil, which provides ample acetyl-CoA to run the CAC in their tissues. One might postulate the need to have some of the acetyl-CoA be derived from fatty acid oxidation.

However, healthy subjects who are on a high-carbohydrate, fat-free diet (Ornish or Pritikin diet) have no muscle weakness. Healthy subjects who are on a high-fat, carbohydrate-free diet (Atkins diet, or the Inuits’ traditional diet of seal and fish) also have no muscle weakness. So, the muscle weakness of the long-chain fat oxidation disorder patients cannot be explained by a decreased availability of acetyl-CoA fuel. Therefore, there must be some decrease in the tissues’ ability to convert acetyl-CoA fuel to usable energy in patients with long-chain fat oxidation defects. Since there is no evidence of a defect in the respiratory chain of these patients, the defect lies probably in the processing of acetyl units by the CAC.

Proper operation of the CAC requires constant anaplerosis to compensate for physiological cataplerosis. When leakage of CAC intermediates is increased by an injury such as ischemia/reperfusion that damages cell membranes, the usual anaplerotic mechanisms can no longer compensate for the increased leakage. Anaplerosis from physiological concentrations of pyruvate (0.05–0.1 mM in plasma and even less within the cell) is a slow process because of the high _K_m of pyruvate carboxylase for pyruvate (0.4 mM). Substantial anaplerosis from pyruvate is observed at supraphysiological pyruvate concentrations (22–24). This explains why adding 1–3 mM pyruvate to the coronary blood improves the contractile ability of the heart (25–27) and decreases infarct size. This also explains why pyruvate improves the function of hearts perfused with AcAc (28). In long-chain fat oxidation disorders, rhabdomyolysis (CPK release in plasma) most likely results also in the leakage of catalytic intermediates of the CAC. When this occurs, it is likely that the rate of operation of the CAC is decreased in spite of ample supply of acetyl-CoA. Indeed, ongoing studies in catheterized pig hearts and in perfused rat hearts show rapid anaplerosis from low concentrations of [U-13C3]propionate, [5,6,7-13C3]heptanoate, and [3,4,5-13C3]BKP, i.e., from precursors of [U-13C3]propionyl-CoA (29). Anaplerosis from these substrates was demonstrated by the formation of M3 mass isotopomers of succinate. These animal data support our hypothesis that the beneficial effect of a dietary treatment of VLCAD deficiency with triheptanoin results from the anaplerotic character of this substrate.

In conclusion, the clinical and biological data of the present investigation, interpreted in the context of our animal studies, strongly support our hypothesis that the impairment in energy production in VLCAD deficiency results from an impairment in the oxidation of acetyl groups in the CAC. This occurs even in the presence of C4 ketone bodies derived from dietary _even_-chain MCT. The administration of heptanoate, a precursor of anaplerotic propionyl-CoA, results in a rapid and profound improvement in the cardiovascular and muscular status of the patient. Heptanoate and its derived C5 ketone bodies provide propionyl-CoA, which replenishes the pool of catalytic intermediates of the CAC. The resulting stimulations of acetyl-CoA oxidation, NADH production, and ATP regeneration improve muscle and heart contraction, restore membrane function, and decrease the loss of CAC intermediates.

The present report opens the way to the treatment with triheptanoin of each of the defects in long-chain fatty acid oxidation in children and adults. We are presently treating and evaluating 22 such patients. The clinical and biochemical data appear very promising in those with deficiencies of CPT I, translocase, CPT II, VLCAD, LCHAD, and trifunctional protein, as was suggested by the in vitro data with odd-carbon fatty acids in fibroblasts from patients with these disorders.