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Chronic complications observed in patients with long-chain 3-hydroxyacylCoA dehydrogenase (LCHAD) or trifunctional protein (TFP) deficiency may be mediated by the accumulation of 3-hydroxy fatty acids or 3-hydroxyacylcarnitines. To understand variation in metabolite accumulation, their concentrations were measured by tandem mass spectrometry before and after a mixed meal and moderate intensity exercise. Subjects who were homozygous or heterozygous for the common mutation (c.1528G>C) in the TFP alpha subunit (LCHAD deficiency) had significantly higher 3-hydroxyacylcarnitines than subjects with TFP deficiency. Feeding a mixed meal significantly suppressed and exercise significantly increased plasma 3-hydroxyacylcarnitines concentrations.
Patients with undiagnosed long-chain 3-hydroxyacylCoA dehydrogenase (LCHAD) and trifunctional protein (TFP) deficiency typically present acutely with nonketotic hypoglycemia, and lactic acidosis that can be complicated by cardiomyopathy and or hepatic encephalopathy 1. The underlying etiology of these acute symptoms appears to be a lack of energy and specifically a lack of energy from carbohydrates. If dietary treatment is initiated immediately, including intravenous dextrose, avoiding prolonged fasting and providing adequate calories, the acute symptoms most often resolve and can be prevented. However, most patients develop some chronic complications such as recurrent rhabdomyolysis, chorioretinopathy with vision loss or peripheral neuropathy with loss of mobility despite dietary treatment 2. The underlying etiology of these chronic complications is not completely understood. One potential mechanism is the toxic effects of accumulating metabolic intermediates, 3-hydroxy fatty acids or their carnitine esters. If the 3-hydroxy fatty acids and hydroxyacylcarnitines are mediating some of the chronic complications of these disorders, then understanding the effects of fasting, feeding and exercise on the accumulation of these metabolites is critical for improving dietary therapy and preventing chronic complications.
Several groups have shown that dietary intake of long-chain fatty acids (LCFA) is directly related to but that medium chain triglyceride (MCT) supplementation is inversely related to fasting hydroxyacylcarnitine concentrations 3, 4. As LCFA intake increases as a percent of total energy, concentration of long-chain hydroxyacylcarnitines increases. Conversely, as MCT intake increases as a percent of total energy, long-chain hydroxyacylcarnitine concentrations decrease. We now further investigate the plasma long-chain 3-hydroxyacylcarnitines responses to feeding and exercise in 8 subjects with LCHAD or TFP.
TFP functions as a heterooctomer; the alpha subunit encodes the hydratase and the LCHAD activities and the beta subunit encodes the thiolase activity but none of functions are active if the holoenzyme doesn’t form. About 80% of the alleles in LCHAD deficiency have a common point mutation, a G to C substitution at nucleotide 1528 of the cDNA within the LCHAD activity domain of the alpha subunit 5. This missense mutation reduces LCHAD activity but does not affect the protein assembly or expression so hydratase and ketothiolase activity are relatively preserved. However, other mutations on both the alpha and beta subunit have been shown to decrease the folding and stability of the complex resulting in loss of all three enzymatic functions, so-called TFP deficiency 6, 7. It could be speculated that patients homozygous for the common mutation would have higher hydratase activity resulting in accumulation of hydroxyacylcarnitines. In contrast, patients with TFP deficiency might have lower hydratase activity and therefore have lower formation of the 3-hydroxy fatty acid and its carnitine ester. We therefore propose there is a genotype/phenotype correlation that is correlated with the 3-hydroxyacylcarnitine concentrations in response to fasting, feeding and exercise.
Subjects ranged in age from 7 to 14 years. Six subjects were female; three were male. The Institutional Review Board at OHSU approved the study protocol, each subject’s legal guardian gave written informed consent and each subject gave written assent.
Subjects were admitted to the Oregon Clinical & Translational Research Institute’s inpatient unit. Prior to the meal and exercise test, an intravenous catheter was placed in a peripheral arm vein to facilitate multiple blood draws.
The metabolic response to a single liquid test meal was measured for each subject as previously described8. The macronutrient content of the liquid meal was 11% protein, 67% carbohydrate, 10% long-chain fat and 12% medium chain triglyceride and the meal provided 500 kcals. Blood samples were drawn immediately before consuming the liquid meal (8–10 hours fasting) and again 1, 2 and 4 hours post-prandially. Plasma samples were analyzed for hydroxyacylcarnitine concentrations.
Each subject completed a moderate intensity exercise test as previously described 9. Briefly, exercise testing was on a treadmill with monitoring of expired gases using a metabolic cart (Sensormedics Corp. model 29n, Yorba Linda, CA). Resting respiratory rate, ECG, blood pressure and temperature were measured prior to and during exercise testing. After a warm-up phase, speed and grade were increased every two minutes until the subject’s heart rate was 60–70% of predicted heart rate maximum (HRmax, 220 – age) 10–12. Subjects achieved their target HR range within the first ten minutes and continued at the same intensity for an additional 30 minutes. Total exercise time was 45 minutes. A blood sample was collected prior to the exercise protocol (pre), after exercise (post), and again after 20 minutes of rest (recovery). Blood was analyzed for plasma acylcarnitines.
Plasma hydroxyacylcarnitine concentrations were measured by tandem mass spectrometry 13. Plasma hydroxyacylcarnitines are tightly correlated to hydroxy fatty acids and serve as a marker of hydroxylated fatty acid species (carnitine esters and free fatty acids) in plasma3. The sum of the long-chain hydroxyacylcarnitine concentrations for each blood sample was calculated and used in the data analysis. Acylcarnitine species included in the calculated total were C14:0-OH, C14:1-OH, C16:0-OH, C16:1-OH, C18:0-OH, C18:1-OH and C18:2-OH. Blood concentrations of measured parameters were plotted over time to create a post-prandial and post-exercise response curve. Total area under the curve was calculated by the trapezoidal method. Differences between the groups were compared by repeated measures analysis of variance using Prism software version 5.0 (GraphPad Software, San Diego, CA).
Subject age, sex, known mutations and TFP enzyme activities are given in Table 1. Enzyme activities were completed in cultured skin fibroblasts. Hydratase activity is not often included in the assay as other cellular hydratases confound the results; LCHAD activity is used to determine alpha subunit function and thiolase activity is used to determine beta subunit function14. The two TFP deficient subjects were siblings. Subjects with TFP deficiency had significantly lower 3-hydroxyacylcarnitines at all time points compared to subjects who were homozygous or heterozygous for the common mutation. However, there was no significant difference in hydroxyacylcarnitines between subjects who were homozygous compared to heterozygous for the common mutation at any time point. A mixed meal containing MCT reduced fasting 3-hydroxyacylcarnitines by approximately 40% in 4 hours among subjects who were homozygous or heterozygous for c.1528G>C (Figure 1). Post-prandial changes in 3-hydroxyacylcarnitines were minimal in subjects with TFP deficiency. Moderate intensity exercise increased 3-hydroxyacylcarnitines by 30% in all subjects (Figure 2). The data suggest subjects with at least one allele containing the common mutation have significantly higher 3-hydroxyacylcarnitines than subjects with TFP deficiency.
The genotype correlation with plasma hydroxyacylcarnitines supports the hypothesis that patients with mutations that preserve the hydratase activity of trifunctional protein produce and accumulate more hydroxyacylcarnitines than patients with mutations that result in a loss of all three enzyme activities. The weakness of this data is that the 2 subjects with TFP deficiency were siblings and this could represent a specific observation in one family that is not observed in other patients with TFP deficiency. Further studies in more patients with TFP are needed to confirm these results. If this observation is confirmed in a larger cohort of TFP deficient patients, then dietary interventions aimed at decreasing plasma hydroxyacylcarnitines might be tailored to the genotype of the patient. Specifically, patients who are homozygous or heterozygous for the common mutation will lower their hydroxyacylcarnitines by consuming frequent low-fat meals supplemented with MCT as currently recommended. The patients may also lower the post-exercise rise in hydroxyacylcarnitines by consuming carbohydrate or MCT prior to that bout of exercise9, 15. In contrast, patients with TFP deficiency may not exhibit much change in post-prandial or post-exercise hydroxyacylcarnitines with these interventions.
However, there is a paucity of evidence that lowering long-chain hydroxyacylcarnitines or their free acids improves long-term outcomes and decreases chronic complications among patients with LCHAD or TFP deficiency. We have demonstrated a strong negative correlation with plasma hydroxyacylcarnitine concentrations and progression of retinopathy in 14 subjects with LCHAD or TFP deficiency 16. In that cohort, subjects with TFP deficiency had lower hydroxyacylcarnitine and mild or no progression of retinopathy over 4 years. Among subjects who were homozygous or heterozygous for the common mutation, there were some subjects with high hydroxyacylcarnitines that had moderate to severe progression of retinopathy during follow up. Conversely, subjects who were homozygous or heterozygous for the common mutation that had lower hydroxyacylcarnitines had mild or no progression of retinopathy over time. The strong correlation between hydroxyacylcarnitines and progression of retinopathy suggests a causative relationship but no direct toxicology data currently exists. Additional long-term follow-up data in patients with LCHAD and TFP deficiency are needed to determine if lowering hydroxyacylcarnitines with dietary interventions decreases the appearance and progression of long-term complications of LCHAD or TFP deficiency.
This study was supported by the National Institute of Diabetes, and Digestive and Kidney Diseases (NIDDK) F32DK065400, and the Oregon Clinical and Translational Research Institute (OCTRI), grant number ULI RR024140 from the National Center for Research Resources (NCRR). This study was approved by the Human Subjects Institutional Review Board at OHSU.