The pathway of mitochondrial fatty acid b-oxidation contributes to energy homeostasis, especially in the heart, the liver and the skeletal muscle. This pathway provides the increased energy demanded by tissues during fasting, febrile illness or increased muscular activity. b-oxidation of long chain fatty acids is a primary source of energy for cardiac muscle. Hepatic b-oxidation provides ketone bodies as fuel for extrahepatic organs, mainly the brain, when the blood glucose levels are low.

During fasting, as carbohydrate reserves fall, the insulin to glucagon ratio decreases. This decreasing ratio of insulin to glucagon increases the rates of hepatic gluconeogenesis and fatty acid oxidation. The breakdown of tissue protein provides amino acids for gluconeogenesis and lipolysis provides a source of fatty acids for fatty acid oxidation. To conserve blood glucose, tissues such as skeletal and cardiac muscle oxidize fatty acids completely within the Krebs cycle without the generation of ketone bodies.

In the fed state, the insulin-to-glucagon ratio is high and the fatty acid oxidation pathway is relatively inactive. This postprandial inactivity of the fatty oxidation pathway contributes to the difficulty of diagnosing patients suspected of having defects in the FAO pathway particularly when the patients are asymptomatic.

(Copyright 1999 by W.B.Saunders Company, reprinted with permission.)

Transport of Fatty Acids into the Cytosol and Activation

Fatty acids are mobilized from adipose tissue and transported in the circulation bound to albumin.

Fatty acids are taken out of the circulation and into the cell by energy-dependent, tissue-specific fatty acid transporter proteins (FATPs) (Fig. 1). Once in the cytosol, nonesterified fatty acids are then bound to fatty acid binding proteins.

Fatty acids are then activated by esterification with coenzyme A to form acylCoA esters. This process requires the activity of acylCoA synthetases.

AcylCoA may:

1. enter the mitochondria for b-oxidation,
2. be incorporated into triglycerides and lipoprotein particles in the liver and gastrointestinal cells,
3. be used in acylation reactions or
4. be incorporated into phospholipids for membrane synthesis.

Entry of acyl groups into mitochondria for b-oxidation during fasting is regulated by the activity of carnitine-palmitoyl transferase I (CPT-1). CPT-1 is the first enzyme of the carnitine shuttle and is located on the outer mitochondrial membrane. It is regulated by the intracellular concentration of malonyl-CoA, a precursor substrate for fatty acid biosynthesis.

The Carnitine Shuttle and Carnitine Transport

Carnitine (g-trimethyl-b-hydroxybutyrate) is required for the transfer of long chain acyl groups into the mitochondria for fatty acid oxidation. Although there is an endogenous biosynthetic pathway for carnitine, most of the carnitine used in the shuttle is dietary in origin. Carnitine is transported into the cytosol by a plasma membrane-bound sodium-dependent carnitine transporter. Two known transporters of carnitine exist in the plasma membrane: one is present in muscle, skin fibroblasts and kidney and another in liver.

The carnitine shuttle facilitates the transfer of acyl groups across the mitochondrial membrane by decreasing the cytosolic concentration of acyl-CoA. Carnitine forms an ester bond with long chain carboxylic acids by the action of CPT-1. Acyl CoA and free carnitine are converted to acylcarnitine and free CoA.

Acylcarnitine species are translocated into the mitochondrial matrix by the inner mitochondrial membrane transporter, carnitine:acylcarnitine translocase. Intramitochondrial free carnitine is exchanged for the acylcarnitine in this process.

Once in the mitochondrial matrix, the acylcarnitine is reconverted to acylCoA and free carnitine by the action of CPT-2, located on the inner aspect of the inner mitochondrial membrane.

Mitochondrial B-Oxidation and Electron Transfer

The acyl-CoA ester enters the pathway of mitochondrial b-oxidation and with each turn of the b-oxidation spiral, the chain length of a saturated acyl-CoA is shortened by two carbon atoms, i.e., an acetylCoA moiety is released. In muscle and heart, acetyl-CoA is completely oxidized in the Krebs cycle to carbon dioxide and water. In liver and kidney, acetylCoA is largely converted to ketone bodies via the HMG-CoA pathway and the ketone bodies are exported for further oxidation in tissues such as brain and muscle.

The b-oxidation pathway consists of repeated cycles of four sequential reactions: acylCoA dehydrogenation, 2-enoyl-CoA-hydration, L-3-hydroxy-acyl-CoA dehydrogenation and 3-ketoacylCoA thiolysis. Each reaction is catalyzed by multiple enzymes with partially overlapping chain length specificity.

These enzymes seem to be organized in two different systems. One is located on the inner mitochondrial membrane and specifically catabolizes long chain acyl-CoAs. The other system, located in the mitochondrial matrix, catabolizes medium and short chain acyl-CoAs. Long chain specific enzymes of the membrane bound system are organized so that substrates are channeled without accumulation of free intermediates.

Inherited Defects of Fatty Acid Oxidation: Clinical Presentation

Inherited defects of almost all the enzymatic steps in this pathway, transmitted as autosomal recessive traits, have been identified in humans. The incidence of each of these disorders is unknown, except for medium chain acyl CoA dehydrogenase (MCAD) deficiency, which is the most frequent fatty acid oxidation disorder. It has an incidence of approximately 1 in 18,000 births in Caucasian populations of northern European extraction. Fatty acid oxidation disorders may account for 5% of episodes of SIDS.

Patients with fatty acid oxidation disorders usually have symptoms related to fasting intolerance. There is often a history of reduced caloric intake, febrile illness or excessive muscular exertion preceding the onset of symptoms. The symptoms may be related to liver dysfunction, muscular dysfunction (cardiac or skeletal) or both. The age of onset ranges from newborn to young adult.

There is considerable phenotypic overlap (Table 1).

A few of these conditions with unique clinical presentations will be covered. Of note, most of these conditions are characterized by recurrent episodes of disease with intervening periods of normality or near normality.

Defects of the Carnitine Cycle

Carnitine transporter defect

The lack of the plasma carnitine transporter results in urinary carnitine wasting. Patients with this condition may present within the first year of life with hypoketotic hypoglycemia, liver failure or a Reye-like syndrome. Older patients may present with a skeletal or cardiac myopathy. Plasma carnitine levels are extremely reduced.

Carnitine Palmitoyl Transferase I Deficiency

The presentation of this condition is triggered by mild viral illness. Affected children may present with hepatomegaly, lethargy and nonketotic hypoglycemia. They have mild hyperammonemia, elevated free fatty acids and elevated liver function tests. In this condition, carnitine levels are normal or increased rather than decreased. Children with severe disease may have developmental delay.

Carnitine-Acylcarnitine Translocase Deficiency

This condition usually presents in the newborn period with seizures, cardiac arrhythmias and apnea. Fasting usually triggers episodes. Patients usually have nonketotic hypoglycemia and marked hyperammonemia. Carnitine levels are usually reduced. Episodes may repeat with cardiac, hepatic and neurologic deterioration.

Carnitine Palmitoyl Transferase II Deficiency

The neonatal presentation causes hepatomegaly, cardiomegaly, cardiac arrhythmia, hypotonia, seizures, renal dysgenesis and dysmorphic features that may resemble Zellweger syndrome. Hypoketotic hypoglycemia, with elevated levels of creatine kinase, and absent dicarboxylic acids are also seen as part of this presentation.

Defects of the B-Oxidation Spiral

Medium Chain Acyl CoA Dehyrogenase (MCAD) Deficiency

The typical presentation for this condition is that of a child presenting between six months and two years of age with acute illness (vomiting and lethargy), following a fasting period of more than 12 hours secondary to an intercurrent illness. There is clinical heterogeneity in MCAD deficiency, even within the same family. SIDS, episodic hypoglycemic coma or recurrent Reye syndrome have all been described in MCAD deficiency. Delays in diagnosis and institution of therapy may place these patients at high risk for long term disability. During episodes, the patients have hypoketotic hypoglycemia, with elevated blood ammonia and liver function tests. The child is asymptomatic between episodes and further episodes can be prevented by providing adequate dietary intake.

Long Dhain Acyl CoA Dehyrogenase (LCHAD) Deficiency

Most patients with this condition will present with fasting-induced hypoketotic hypoglycemia, after a long fast caused by an intercurrent infectious illness. During these episodes, CK levels are usually elevated. Phenotypic heterogeneity also exists in this condition. A few patients have presented with cardiomyopathy (often fatal) or with a more indolent limb-girdle myopathic presentation. The condition also may present with exercise-induced rhabdomyolysis. Hepatic dysfunction can be a frequent finding. In occasional patients, massive total hepatic necrosis is found. The progressive sensorimotor peripheral neuropathy and pigmentary retinopathy found in a few patients are not characteristic of mitochondrial fatty acid oxidation defects. The acidosis is more prominent than in other fatty acid oxidation disorders and the lactic acidosis noted in acute episodes is virtually unheard of in other defects of this pathway.

Very Long Chain Acyl CoA Dehyrdogenase (VLCAD) Deficiency

Most patients suffer from the severe cardiomyopathic form with early onset and poor outcome. These patients have hypertrophic cardiomyopathy, and high morbidity and mortality. A milder form may be reminiscent of MCAD deficiency, presenting with hypoketotic hypoglycemia. A third form may present with exercise-induced rhabdomyolysis or stress associated with viral illness.

Short Chain Acyl CoA Dehyrdogenase (SCAD) Deficiency

Most patients have presented in the neonatal period with a variable phenotype including metabolic acidosis, failure to thrive, developmental delay, myopathy and seizures. Hypoketotic hypoglycemia has not been described associated with this condition. It should also be considered in the differential diagnosis of progressive external ophthalmoplegia and congenital multicore myopathy.

Multiple Long Chain Acyl CoA Dehyrdogenase (MAD) Deficiency

It is characterized, clinically, by hypoketotic hypoglycemia and metabolic acidosis. Complete deficiency of electron transfer flavoprotein-ubiquinone oxidoreductase is also associated with dysmorphic features resembling Zellweger syndrome, dysgenesis of the kidneys and brain. Most patients with severe disease do not survive the first few weeks of life.

Laboratory Diagnosis

For most disorders, the diagnosis is more direct when appropriate blood and urine samples are collected during a period of acute metabolic decompensation. Because correction of the hypoglycemia often corrects the metabolic decompensation and switches off the endocrine-mediated flux of substrate for fatty acid oxidation, the production of abnormal metabolites rapidly declines.

Investigation in the Acute Phase

Blood

• Gases, electrolytes
• Glucose will be low (often undetectable).
• Transaminases will be modestly elevated (3x normal)
• Ammonia will be modestly elevated (100-300 micromoles/l), except in the translocase defect in which it may be up to 800 µmol/l.
• Lactate, pyruvate
• CK may be elevated in myopathic presentation (up to 10,000x normal) especially in CPT-2 deficiency, the myopathic form of VLCAD deficiency, and LCHAD deficiency.

Urine

• Ketones will be absent or low.

Urine

• Ketones will be absent or low.

Specialized Laboratory Investigations

The differential diagnosis of fatty acid oxidation disorders is an increasingly complex process. Complete work-up requires the performance of multiple analyses.

Urine organic acid analysis: During illness, these disorders are associated with inappropriate dicarboxylic aciduria in which urinary medium chain dicarboxylic acids are elevated, while urinary ketones are not. In the following disorders, specific abnormalities are present: MCAD, VLCAD, SCAD, LCHAD and SCHAD deficiencies. CPT-2 deficiency will show nonspecific dicarboxylic aciduria. Urine organic acids may be normal when the patients are asymptomatic (except for MCAD deficiency).

Urine acylglycines: have enough specificity and sensitivity to diagnose MCAD deficiency even when patients are well. Other condition in which this analysis has been useful is glutaric aciduria type II.

Total serum carnitine levels: can be reduced in many of these disorders, except for CPT-1 deficiency in which it can be elevated. In primary carnitine deficiency, the levels are severely decreased (<5% of normal). Urine carnitine is only useful in primary carnitine deficiency in which there are big losses of carnitine in the urine.

Serum acyl carnitine profile: will show specific abnormalities in the acute phase for the following defects: translocase, CPT-2, VLCAD, LCHAD, MCAD, SCAD and SCHAD deficiencies. The ratios of acylcarnitine to free carnitine will be elevated. An abnormal profile indicates the possibility of a FAOD but it is not sufficient to confirm the diagnosis. A normal profile does not rule out these conditions when the sample is collected from asymptomatic patients.

Analysis of free fatty acids and 3-hydroxy fatty acids in plasma: a high free fatty acid to ketone bodies ratio is suggestive of a long chain fatty acid oxidation defect (i.e., LCHAD deficiency)

Investigation in the Chronic Phase

For a precise diagnosis, and if no revealing information is obtained with the investigations performed in the acute phase, the following tests should be performed:

Prolonged fasting test under strict medical supervision, taking blood samples at regular intervals to measure glucose, ketone bodies and free fatty acids. An acylcarnitine profile can be obtained at the same time.

Metabolic flux study performed in cultured cells (fibroblasts). This involves monitoring the rate of production of radioactive end products of fatty acid oxidation (H2O or CO2) for radiolabeled precursor fatty acids or detection of accumulated metabolites resulting from an enzyme defect.

In vitro analysis, probing the fatty acid oxidation pathway. This method is based on measurement by tandem mass spectrometry of disease-specific acylcarnitines produced when stable isotope fatty acid precursors are incubated with cells in the presence of excess of L-carnitine. This represents a single test for all the enzymes from translocase to SCAD. This technique is highly specific and can be applied for prenatal diagnosis.

Enzyme assay: in fibroblasts or other tissue, such as muscle or liver. Because of the frequent finding of overlapping chain-length specificities, complex analysis, using a mixture of different chain-length substrates and immunoprecipitation with antibodies to the different enzymes, is required.

Carnitine transport assay: specific for systemic carnitine deficiency.

Molecular analysis: the genes for most of the enzymes of FAO have been identified, and mutation analysis is available for a number of genes: CPT-I, CPT-II, VLCAD, TFP, MCAD and others. Prevalent mutations have been identified in patients with MCAD deficiency (A985G) which has led to a PCR based assay. In LCHAD deficiency there is a common mutation (G1528C) that has led to a PCR based analysis. In CPT-II deficiency, a C439T mutation accounts for 60% of mutations in patients with CPT-II deficiency of adult onset. On the other hand, disorders such as VLCAD deficiency are genetically heterogeneous.

Prenatal Diagnosis

Prenatal diagnosis of fatty acid oxidation disorders in chorionic villous material, as well as amniocytes, since all the enzymes of mitochondrial fatty acid oxidation are expressed in these cell types. Acylcarnitine profile or direct enzyme assays can be performed. If the molecular defect (MCAD, for example) has been determined in the proband, molecular analyses can be done.

Treatment

Medical Management of the Acute Episode

1. I.V. fluids, glucose ---> 10% dextrose at rates of 10 mg/kg/min or greater to achieve normal glucose concentrations. Basing the delivery of carbohydrate on blood glucose level alone may underestimate the carbohydrate demand, as tissues will be depleted of glycogen reserves.
2. I. V. carnitine restores tissue carnitine concentrations for the transport of fatty acids in the mitochondria. This treatment also provides adequate supply of coenzyme A and removes toxic metabolites in the form of carnitine adducts that are readily excreted in the urine.
3. Pharmacological support (cardiomyopathy).

Long Term Management of Asymptomatic Patients

For prophylactic treatment, there have been no controlled, prospective studies of long term therapeutic management of patient with FAO defects.

Diet, Supplements and Drugs

Diet

• avoid fasting with frequent feedings and increase carbohydrate calories during periods of high caloric demand
• low fat diet, high carbohydrate diet (30-35% calories from fat)
• medium-chain triglyceride supplementation in long-chain fatty acid defects.
• use of uncooked cornstarch at bedtime to prevent early morning hypoglycemia after the overnight fast.
• essential fatty acids (i.e., linoleic and linolenic acids) supplementation to prevent growth restriction and dermatitis that are seen with their deficiency.

Supplements

• carnitine supplementation to maintain normal plasma free or total carnitine, or to enhance excretion of abnormal metabolites even if plasma carnitine levels are normal. Carnitine supplementation facilitates excretion of accumulated toxic intermediates as carnitine conjugates and thereby restores the acylCoA/free CoA ratio.
• glycine can be used to condense with organic acids excreted in multiple acyl-CoA dehydrogenase deficiency (glutaric aciduria type II) to form acylglycine esters and promote their excretion.
• riboflavin is a cofactor of the electron transfer flavoprotein and is used in some cases of multiple acyl-CoA dehydrogenase deficiency (glutaric aciduria type II). It is used as a cofactor in cases of short chain acyl-CoA dehydrogenase (SCAD) deficiency.
• Because docosahexaenoic acid depletion has been observed in patients with LCHAD deficiency and it is thought to cause the pigmentary retinopathy seen in this condition, its supplementation is currently recommended.

Drugs

• prednisone can be used to treat patients with LCHAD deficiency experiencing myopathy or patients with VLCAD deficiency who experience recurrent myoglobinuria.