Fatty Acid Oxidation Defects

Question 1

What are Fatty Acids?

Answer 1

• Fatty acids are a major dietary ingredient and an excellent source of energy
• Most dietary fat consists of triglycerides containing long-chain fatty acids
• The energy obtained from excess calorie intake is used for the biosynthesis of long chain fatty acids and storage as fat. Body fat is an energy dense material
• Fat is stored in adipose tissue in the form of long-chain triglycerides

Question 2

What are functions of Fatty Acids?

Answer 2

Fatty acids can be readily mobilised in the non-fed state

• Production of ketones by hepatic β-oxidation
• Give a high yield of ATP through fatty acid oxidation and oxidative phosphorylation

They are preferentially used by some tissues as a major energy source

• Heart muscle derives 60% of its energy from long-chain fatty acid oxidation

Skeletal muscle uses fatty acid oxidation at rest but especially during extended aerobic muscle exercise

Question 3

What are features of Ketone Bodies?

Answer 3

Liver only tissue to produce ketone bodies

• 3-hydroxybutyrate, acetoacetate and acetone

Liver unable to utilise ketone bodies (export only)

Ketone bodies are major fuel for peripheral tissues during fasting

Ketones “spare” glucose for the brain and other vital tissues

In prolonged fasting ketones provide >80% bodily energy

Question 4

What are the effects of Fatty Acid Defects?

Answer 4

Usually present in infancy/early childhood often preceded by illness / poor food intake

Fatty acid oxidation defects lead to a reduced or absent ability to produce ketones leading to hypoketotic hypoglycaemia, encephalopathy, coma and death. There are also adverse effects in tissues and organs that preferentially use fatty acids for energy:

• In skeletal muscle - muscle weakness, pain and rhabdomyolysis.
• In heart muscle - cardiomyopathy and conduction defects.
• Liver function can also be compromised leading to abnormal liver function tests.

Question 5

What are the effects of Short Term Fasting?

Answer 5

• In the non-fed state fatty acids are mobilised from adipose tissue (In neonates and infants this may occur after only a few hours, In adults it occurs from 6 to 12 hours post feeding)
• They are transported to the liver as free fatty acids (FFA) and undergo beta-oxidation in the liver to produce ketone bodies
• Ketones are used by extra-hepatic tissues during fasting, although the brain has only a limited capacity to use them for energy

Question 6

How are ketones beneficial in neonates?

Answer 6

• Neonates and infants have an increased head/body ratio as compared to adults and therefore they have a relatively high cerebral glucose requirement.
• They also have a small liver and hence relatively low glycogen stores.
• Without the “glucose sparing” effect of ketones neonates and infants would rapidly develop hypoglycaemia.

Question 7

What are effects of Long term fasting?

Answer 7

• On long term fasting (e.g. starvation) the body adapts by increasing use of body fat and muscle and the brain slowly switches from using glucose as its main energy source to using ketone bodies.
• Over 80% of energy requirements are met by fatty acids and muscle is slowly broken down to provide gluconeogenic substrates as some glucose is still needed.

Question 8

What is the process for Fatty Acid Oxidation?

Answer 8

• Long chain free fatty acids enter the mitochondria as fatty acyl-CoA derivatives via the carnitine shuttle
• Short and medium chain fatty acids enter the mitochondria independently
• During ß-oxidation the fatty acyl-CoA’s are sequentially shortened by two carbon units for each turn of the cycle with the production of acetyl-CoA
• Some of the reducing equivalents (NADH, FADH) produced during ß-oxidation are fed directly into the electron transport chain (ETC) to produce ATP
• Acetyl-CoA is also fed into the citric acid cycle to generate more reducing equivalents and subsequently ATP in the ETC
• In the liver ketones are synthesised from acetyl-CoA for export to peripheral tissues for further oxidation and ATP production

Question 9

How does Hypoketotic Hypoglycaemia occur?

Answer 9

• Due to Impaired ketogenesis leading to production of toxic metabolites
• This lead to sequestration of CoA and carnitine and inhibition of gluconeogenesis / intermediary metabolism

Question 10

When should a fatty oxidation defect be suspected?

Answer 10

Intermittent hypoglycaemia ± acidosis

• episodic encephalopathy / Reye-like episodes
• Failure to thrive / hypotonia / poor feeding

Cardiomyopathy ± liver dysfunction

Exercise/viral induced myalgia / rhabdomyolysis

• Children - myalgia /exercise intolerance
• Adolescents / adults - episodic rhabdomyolysis

Question 11

What are types of Fatty Acid Oxidation Defects?

Answer 11

• Medium chain acyl-CoA dehydrogenase (MCAD) deficiency (Most common)
• Carnitine cycle defects
• B oxidation defects
• Electron Transfer Defects
• Defects of Riboflavin Transport and metabolism

Question 12

What is the enzymatic defect in Primary Carnitine Deficiency?

Answer 12

• The organic cation/carnitine transport OCTN2 is responsible for carnitine uptake across the plasma membrane, particularly in heart, muscle and kidney.
• Defects lead to primary carnitine deficiency with increased renal loss of carnitine, low plasma concentrations and sufficiently low intracellular concentrations to impair fatty acid oxidation.

Question 13

What is the clincial presentation of Primary Carnitine Deficiency?

Answer 13

• Infants: Hypoglycaemia, Liver Dysfucntion, Hyperammonaemia
• Children: Heart failure due to cardiomyopathy, often accompanied by skeletal muscle weakness.
• Adults may suffer fatigue or arrhythmias.
• Many patients remain asymptomatic

Question 14

What is the diagnosis and treatment of Primary Carnitine Disorders?

Answer 14

Diagnosis:

• Very low free carnitine concentration in plasma

Treatment:

• Carnitine replacement, 100 mg/kg/day
• Outcome is Excellent with treatment

Question 15

What is the enzymatic defect in Carnitine Palmitoyl Transferase Deficiency Type 2 (CPT2)?

Answer 15

Carnitine Palmitoyltransferase type 2

Question 16

What are clinical features of Carnitine Palmitoyl Transferase Deficiency Type 2 (CPT2)?

Answer 16

• The severe neonatal form is often fatal and presents with hypoketotic hypoglycaemia, cardiomyopathy, hypotonia and congenital abnormalities.
• There is a milder form often presenting in older children or adults with recurrent attacks of rhabdomyolysis trigged by prolonged aerobic exercise or sometimes catabolic stress.

Question 17

How is diagnosis of Carnitine Palmitoyl Transferase Deficiency Type 2 (CPT2) made?

Answer 17

• In the severe form the plasma acylcarnitines show increased long chain acylcarnitines with free carnitine depletion and fatty acid flux studies show reduced long chain fatty acid oxidation.
• In the mild adult forms acylcarnitines and fatty acid flux studies may be normal.
• However specific enzyme assay of CPT2 will diagnose all forms

Question 18

How is Carnitine Palmitoyl Transferase Deficiency Type 2 (CPT2) managed?

Answer 18

• In the neonatal/infantile form treatment is by a low long-chain fat / high carbohydrate diet supplemented with medium chain triglycerides.
• It is important to avoid fasting and the patient needs an emergency regime during times of catabolism/infection/stress.
• In the adult forms medium chain triglyceride rich meals and maintenance of glycogen stores prior to exercise may help.

Question 19

What is the outcome of Carnitine Palmitoyl Transferase Deficiency Type 2 (CPT2)?

Answer 19

• In the neonatal form there is high morbidity and mortality.
• In the adult form with repeated rhabdomyolysis attacks on exercise there is a risk of renal failure secondary to the rhabdomyolysis.

Question 20

What is the enzymatic defect in MCADD?

Answer 20

Deficiency of Medium chain acyl-CoA dehydrogenase which coverts fat into energy.

• Medium chain fats are 6 to 12 carbon units long
• Partially broken-down fatty acids accumulate in the blood of patients with MCADD.
• C8 (octanoyl carnitine) increased in blood, and is unique to medium chain fatty acids
• Fatty acids are an important energy reserve during periods of poor calorie intake, fasting or during infections

Question 21

How does MCADD develop in neonates?

Answer 21

In babies, there is store of fat from the third trimester but in MCADD, this store of energy is not effectively used and if they are not feeding well, the condition can present clinically

Question 22

What are some genetic factors about MCADD?

Answer 22

• Autosomal Recessive, MCADD gene (ACADM) found on chromosome 1
• Most common mutation is 985A>G (K304E) accounts for approximately 80-85% of clinically presenting disease
• Milder variants picked up by newborn screening
• It is thought that between 1 in 40 and 1 in 80 healthy people are carriers and do not have any symptoms

Question 23

What is the clinical presentation of MCADD?

Answer 23

Well until decompensation (metabolic stress)

• Neonatal period (onset of breast feeding)
• Fasting
• Inter-current infection esp. gastrointestinal

Main feature is Hypoketotic Hypoglycaemia which can cause sudden death or may progress to Reye’s Syndrome.

Onset tends to be more commonly from 2 months to 4 years of age but may occur, rarely, at any time even in adulthood.

Muscle and liver problems are not a major feature of this disorder.

Sometimes there is significant ketosis.

Mean age at presentation 13/12 months

Seasonal (Autumn / Winter)

20-25% mortality, 30% survivors CNS damage

Question 24

How is MCADD initially screened?

Answer 24

1. Initial C8 (octanoyl carnitine) value > 0.4µM
2. Repeat in duplicate and measure C10
3. Average of all three C8 values > 0.5µM and C8/C10 ratio is >1.0 referred for clinical follow up
   
   • Ratio helps distinguish between MCADD and other (rare) causes of increased C8
   • Stressed heterozygotes, GA2 (MADD), ketosis, dead spiders…

Question 25

What happens with the postive screens of MCADD?

Answer 25

• All presumptive positives should be referred on the same day that the final screening result has become available.
• The first clinic appointment should take place within 24hrs of the final result becoming available
• A confirmatory acyl carnitine scan should have been undertaken and reported to the MCADD designated team – if possible in time for the clinic appointment.

Question 26

How is diagnosis of MCADD confirmed?

Answer 26

1. Urine sample for organic acid analysis
   
   • Characteristic peaks of hexanoylglycine and suberylglycine
   • Increased medium chain dicarboxylic acids
2. Acylcarnitines
   
   • Increased C6, C8, C10:1 and C10
3. Increased ratios of free fatty acids/3-hydroxybutyrate
4. Fibroblast fatty acid oxidation of [9,10]-3H myristate is reduced
5. Dried bloodspots sent for DNA analysis
   
   • First line is screening for common variant (985A>G)
   • If one or no copies found, gene sequenced
6. Interpretation may be difficult. Pathogenicity of newly identified variants may not be known

Question 27

What is the treatment of MCADD?

Answer 27

1. Avoidance of fasting – chocolate, crisps, non-diet drinks, milk (Safe length of fast increases with age)
2. Emergency regimen for intercurrent illness. Based on glucose polymer drinks
   
   • Information leaflets for parents and professionals when attending hospital (A&E)

Question 28

What is the outcome of disease in MCADD?

Answer 28

• If untreated there is a high morbidity and mortality associated with the hypoglycaemia.
• Treatment is by avoidance of fasting and is usually very successful.

Question 29

How is family counselling done in MCADD?

Answer 29

1. Genetic counselling of families: 1 in 4 (unless parent affected!)
2. Older siblings born before newborn screening started will need to be tested biochemically– arranged by metabolic clinician
3. Next child: early testing at 24-48 hrs as well as routine screening test
   
   • Vitally important to treat new sibling as high risk of MCADD until proven otherwise
   • Metabolic team should be involved and strict protocols regarding feeding adhered to

Question 30

What is the enzymatic defect in Very Long Chain Acyl-CoA Dehydrogenase Deficiency?

Answer 30

Very long chain acyl-CoA dehydrogenase deficiency ACADVL

Question 31

What is the clinical presentation of Very Long Chain Acyl-CoA Dehydrogenase Deficiency?

Answer 31

• In its severest form this disorder can present with collapse and death in the neonatal period with acidosis and heart and liver disease.
• Less severe forms may show failure to thrive from an early age with repeated attacks of hypoglycaemia often triggered by intercurrent infections.
• There may be liver disease & cardiomyopathy.
• There is also a much milder adolescent/adult onset forms with muscle weakness or exercise intolerance with rhabdomyolysis on prolonged exercise.

Question 32

How is diagnosis of Very Long Chain Acyl-CoA Dehydrogenase Deficiency made?

Answer 32

• Increased ratios of free fatty acid/3-hydroxybutyrate
• Urine organic acid analysis shows increased excretion of dicarboxylic acids
• Plasma acylcarnitines show increased long chain acylcarnitines particularly C14:1, C16:1, C14, C16 & C18:1.
• Fatty acid flux studies in fibroblasts show reduced long chain fatty acid oxidation

Question 33

How is Very Long Chain Acyl-CoA Dehydrogenase Deficiency managed?

Answer 33

• The severe hypoglycaemia is treated with high carbohydrate feeds often delivered overnight by nasogastric tube.
• Long chain fats need to be avoided.
• To provide energy medium chain triglycerides are used as these bypass the enzyme block and provide energy predominantly through ketone body production in the liver.
• Important to avoid a catabolic state.

Question 34

What is the outcome of Very Long Chain Acyl-CoA Dehydrogenase Deficiency if untreated?

Answer 34

• If untreated, the severest form (i.e. infantile cardiomyopathy) will have a high morbidity and mortality.
• However for the moderately severe, infantile/milder phenotypes with treatment there is generally a good outcome and often it may be very good.

Question 35

What is the enzymatic defect in Multiple Acyl-CoA Dehydrogenase deficiency (MADD) (aka GA2)?

Answer 35

• Electron transfer flavoprotein (ETF) and ETF ubiquinone oxidoreductase carry electrons to the respiratory chain from multiple FAD linked dehydrogenases.
• Thus defects of ETF or ETFQO lead to multiple acyl-CoA dehydrogenase deficiency. Can also results from defects of riboflavin transport or metabolism.

Question 36

What is the clinical presentation of Multiple Acyl-CoA Dehydrogenase deficiency (MADD) (aka GA2)?

Answer 36

Wide range of clinical severity.

• Severely affected patients present in the first few days of life with hypoglycaemia, heperammonaemia and acidosis. Some patients have congenital abnormalities including large cystic kidneys, neuronal migration defects and facial dysmorphism.
• Less severe cases can present at any age from infancy to adulthood with hypoglycaemia, liver dysfunction and weakness.
• Cardiomyopathy is common in infants.
• Muscle weakness is the most common presentation in adolescents and adults.

Question 37

How is diagnosis of Multiple Acyl-CoA Dehydrogenase deficiency (MADD) (aka GA2) made?

Answer 37

• Increased C6, C8, C10 and C12 on plasma acylcarnitines.
• Increased concentration of a number of pathognomic metabolites on urinary organic acid analysis.

Question 38

How is Multiple Acyl-CoA Dehydrogenase deficiency (MADD) (aka GA2) managed?

Answer 38

• Avoidance of prolonged fasting.
• Treatment with 3 hydroxybutyrate has been used in severe deficiencies.
• Many of the milder defects respond to riboflavin.

Question 39

Whhat are metabolic and detailed testing for fatty acid oxidation defects??

Answer 39

For the metabolic laboratory the first line tests would be:

• measurement of the ratio of plasma free fatty acids to beta-hydroxybutyrate
• urine organic acid analysis by gas chromatography mass spectrometry
• plasma acylcarnitine analysis by tandem mass spectrometry

More detailed testing would include:

• Measurement of the flux of fatty acid oxidation in cultured fibroblasts using radioisotopic methods
• Specific assays for individual enzymes
• Mutation analysis for common mutations. In some cases, full mutation screening

Question 40

When should urine and plasma samples be taken for testing?

Answer 40

It is very important to collect urine and plasma samples when the child is hypoglycaemic as in some cases the abnormalities may disappear once glucose concentrations have been normalised.

Question 41

Who should be tested for Fatty Acid Oxidation Defects?

Answer 41

• A fatty acid oxidation defect should be considered in any child with unexplained hypoglycaemia.
• The fact that the child may be ketotic does NOT exclude this group of disorders.

Question 42

What are the investigations on acute presentation for fatty acid oxidation defects?

Answer 42

• Fluoride sample for Intermediary metabolites glucose, lactate, free fatty acids and 3-hydroxybutyrate
• *Li/hep for insulin, cortisol, growth hormone, amino acids and acylcarnitine profile
• First urine passed for Organic acids (and possibly toxicology)
• Also often useful – LFT’s, CK, blood gases, ammonia
• Only after the *blood samples have been taken is oral or IV glucose given

Question 43

How should confirmation and diffferentiation of fatty acid oxidation defects be made?

Answer 43

By Organic Acids:

• There may be excretion of specific pathognomic metabolites eg hexanoylglycine, suberylglycine and phenylpropionylglycine in MCAD (medium chain AcylCoA dehydrogenase) deficiency.
• OR marked excretion of dicarboxylic and /or 3-hydroxydicarboxylic acids strongly suggestive of a fatty acid oxidation disorder but individual disorders cannot easily be distinguished from one another

Plasma or DBS acylcarnitines may also strongly suggest the diagnosis in some cases.

Final confirmation usually by fibroblast assay, fatty acid oxidation flux or specific enzyme assay and/or mutation analysis

Question 44

What is the use of Intermediary Metabolities?

Answer 44

• Free fatty acids/ 3-hydroxy butyrate.
• A ratio >2 is indicative of a fatty acid oxidation defect. Fatty acids are mobilised but not converted to ketones.
• This test is really only useful when the sample is taken during a period of hypoglycaemia or fasting.

Question 45

What is the use of Organic Acid Profile?

Answer 45

• Urine organic acids are extracted and converted to their TMS esters and detected by GC-MS.
• Specific organic acids and glycine conjugates will be present in urine from patients with fatty acid oxidation defects.
• Note these abnormalities are often only seen during crisis.

Question 46

What is the use of Acyl Carnitine Profiles?

Answer 46

• Plasma and blood spot acylcarnitines are derivatised and detected by tandem mass spectrometry.
• Specific acylcarnitine species will be present or increased in some disorders of fatty acid oxidation.
• Not all disorders have abnormal acylcarnitine profiles and some only have subtle changes.

Question 47

What are Fatty Acid Oxidation flux studies?

Answer 47

• Cultured fibroblasts are incubated in multi-well plates with labelled fatty acids in a buffer – usually [9,10-3H]myristate, palmitate or oleate.
• The fatty acids are transported into the mitochondria & beta-oxidised & the 3H converted to 3H2O. The released labelled water equilibrates within the cell & in turn with the incubation buffer.
• When the buffer is removed after 2 hours incubation it contains the released 3H2O as well as the un-metabolised fatty acid.
• The Released label ( 3H20) is separated from un-metabolised fatty acid using an ion exchange column and counted in a scintillation counter.
• The amount of label released is expressed relative to the amount of fibroblast protein/ well.
• This value will give a measure of the patients ability to carry out fatty acid oxidation.
• Several substrates can be used which will require different chain-length specific enzymes to metabolise them.

Question 48

What are the uses of Specific Enzyme Studies?

Answer 48

• Sonicated fibroblasts are incubated with a specifically labelled compound.
• A product is formed by the enzyme that will incorporate the label. The old labelled compound is separated and discarded.
• The amount of new compound formed is counted in a scintillation counter.
• This value will give a measure of the specific enzyme activity for that patient as nmol product /mg fibroblast protein/min

Question 49

What are the uses of Mutation Analysis?

Answer 49

Has some limitations but useful when a common mutation exists. Some examples:

• MCAD (common K304E)
• VLCAD (no common mutation)
• LCHAD (common E474Q (~ 85%))
• Trifunctional Protein Deficiency presents in same way and has no common mutation
• CPT I no common mutation
• CPT II S113L accounts for ~50% of “mild myopathic” disease