Acylcarnitine Profile

The acylcarnitine profile measures carnitine and its fatty acid and organic acid esters (acylcarnitines) by tandem mass spectrometry in plasma, serum or a dried blood spot.

Carnitine carries long chain fatty acids into mitochondria for beta oxidation and buffers excess acyl-CoA groups, so a block in fatty acid oxidation or in organic acid catabolism causes accumulation of the specific acylcarnitine upstream of the defect.

The profile screens for and helps diagnose more than 30 inborn errors: fatty acid oxidation defects (medium chain, very-long-chain, long chain 3-hydroxyacyl, carnitine palmitoyltransferase and translocase deficiencies, multiple acyl-CoA dehydrogenase deficiency), organic acidaemias (propionic, methylmalonic, isovaleric, glutaric type 1, 3-methylcrotonyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA lyase deficiencies) and the primary carnitine uptake defect.

Chain length is written as the number of carbon atoms (C8 is octanoylcarnitine, C16 is palmitoylcarnitine), with suffixes for hydroxy (OH), dicarboxylic (DC) and double bonds (:1).

Fatty acid oxidation defects together affect about 1 in 10 000 to 1 in 15 000 births, medium chain acyl-CoA dehydrogenase deficiency being commonest (1 in 10 000 to 20 000 in northern Europeans, much rarer in people of African and Asian ancestry). Organic acidaemias affect about 1 in 20 000 to 50 000.

Untreated, fatty acid oxidation defects cause hypoketotic hypoglycaemia, Reye like illness, cardiomyopathy, rhabdomyolysis and sudden infant death; organic acidaemias cause neonatal metabolic acidosis, hyperammonaemia and encephalopathy.

Early diagnosis prevents most deaths and disability, and the profile is a core component of newborn screening and of the investigation of unexplained hypoglycaemia, encephalopathy, cardiomyopathy and myopathy at any age.

Indications. Newborn screening (dried blood spot at 24 to 72 hours). Hypoketotic or inappropriately low ketone hypoglycaemia. Unexplained acute encephalopathy, hyperammonaemia or metabolic acidosis with raised anion gap. Cardiomyopathy or arrhythmia in infancy. Recurrent rhabdomyolysis or exercise intolerance.

Sudden unexplained death in a child (post-mortem blood spot or bile spot). Elevated screening result needing confirmation. Monitoring of treatment (diet, carnitine supplementation). Family members of an affected patient. Not as a general screening test in adults without a metabolic presentation.

Specimen. Plasma or serum (0.5 mL, heparin or EDTA, frozen quickly) or dried blood spot on filter paper. Ideally collected during the acute illness or fasting stress, because interictal profiles can be normal or only mildly abnormal, particularly in long chain defects.

Record time since last feed, fasting duration, glucose, intravenous glucose or lipid infusion, carnitine supplementation and medication (valproate, pivalate containing antibiotics). Send with a urine sample for organic acids taken at the same time. Post-mortem: blood spot or bile spot on filter paper.

Reference values. Laboratory and age specific, in micromol/L. Free carnitine (C0) about 10 to 60 in plasma (newborn blood spot 10 to 50). Acetylcarnitine (C2) 3 to 15. Propionylcarnitine (C3) under 3.

Octanoylcarnitine (C8) under 0.3, decanoylcarnitine (C10) under 0.5, tetradecenoylcarnitine (C14:1) under 0.2, palmitoylcarnitine (C16) under 6, hydroxypalmitoylcarnitine (C16OH) under 0.1, and isovalerylcarnitine (C5) under 0.5. Ratios (C3/C2, C8/C10, C0/(C16+C18)) are interpreted with the laboratory's cut offs.

Interpretation.

• Medium chain acyl-CoA dehydrogenase deficiency: raised C8 (often over 1), with raised C6 and C10:1 and a C8/C10 ratio over 5. Confirmation by ACADM genotype (c.985A>G common in Europeans).
• Very-long-chain acyl-CoA dehydrogenase deficiency: raised C14:1 with C14, C14:2 and C16.
• Long chain 3-hydroxyacyl-CoA dehydrogenase and trifunctional protein deficiency: raised C16OH, C18OH and C18:1OH, associated with maternal acute fatty liver and HELLP in pregnancy with an affected foetus.
• Carnitine palmitoyltransferase 2 and translocase deficiencies: raised C16 and C18:1 with low C0.
• Carnitine palmitoyltransferase 1 deficiency: high C0 with low long chain acylcarnitines (ratio of C0 to C16 plus C18 over 100).
• Multiple acyl-CoA dehydrogenase deficiency (glutaric aciduria type 2): raised C4 to C18 acylcarnitines of every chain length.
• Short chain acyl-CoA dehydrogenase deficiency: raised C4, which is often a benign variant. Isobutyryl-CoA dehydrogenase deficiency also raises C4.
• Propionic and methylmalonic acidaemias: raised C3 with high C3/C2 ratio (cobalamin defects also raise homocysteine).
• Isovaleric acidaemia: raised C5. Beta ketothiolase deficiency: raised C5:1 and C5OH. 3-methylcrotonyl-CoA carboxylase deficiency and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency: raised C5OH. Glutaric aciduria type 1: raised C5DC (glutarylcarnitine); low excretors can have a normal profile, so urine organic acids are also needed.
• Primary carnitine deficiency (carnitine transporter defect): very low C0 (under 10 micromol/L in plasma, under 5 in neonatal blood spot) with low acylcarnitines of every chain, cardiomyopathy and hypoglycaemia. A low C0 in a newborn may reflect an unrecognised maternal carnitine transporter defect, so the mother is also tested.
• Secondary carnitine deficiency occurs with valproate, pivalate containing antibiotics, renal Fanconi syndrome, haemodialysis, organic acidaemias, malnutrition, and total parenteral nutrition without carnitine.
• Mild non-specific elevations occur in fasting, ketosis, prematurity, hepatic disease, and infants fed medium chain triglyceride formula (C8 and C10 raised).

Critical values and findings needing immediate action.

• Raised C8 with C6 and C10:1, or any long chain profile, in a newborn or sick child: avoid fasting, give an emergency regimen of intravenous 10% dextrose, check glucose, ammonia, lactate, ketones, creatine kinase and liver tests, and contact the metabolic team the same day.
• Raised C3 or C5 with acidosis, hyperammonaemia, vomiting or lethargy: metabolic emergency, stop protein intake, give glucose with insulin as needed, ammonia scavengers and early dialysis for ammonia over 400 micromol/L.
• Raised long chain hydroxyacylcarnitines in a newborn: risk of cardiomyopathy, arrhythmia and rhabdomyolysis; avoid long chain fats and give medium chain triglyceride under specialist direction.
• Very low free carnitine with cardiomyopathy: start high dose oral carnitine promptly.
• Abnormal profile in post-mortem blood spot: inform the family and arrange screening of siblings and parents.

Interferences and pitfalls.

• Interictal and well state samples may be normal or near normal in fatty acid oxidation defects (particularly long chain and carnitine palmitoyltransferase 2 defects), so a normal profile does not exclude disease if the clinical picture is compelling; repeat during fasting or illness, or test urine organic acids and DNA.
• Recent carnitine therapy, medium chain triglyceride feeds, intravenous lipid, valproate, pivalate antibiotics, and parenteral nutrition change the pattern and produce false positives (C5 from pivalate, C8 and C10 from medium chain triglyceride).
• Preterm and early sampling (under 24 hours) give high false positive rates; transfused blood dilutes the abnormal pattern.
• Dried blood spot quality (layering, contamination, delay in drying) affects results; plasma is preferred for diagnostic work.
• Different laboratories use different cut offs and instruments, and ratios outperform single analytes.
• Isobaric compounds (for example C5 species, hydroxy and dicarboxylic acylcarnitines, and species with the same mass) cannot be separated without chromatography, and second tier testing reduces false positives.
• Newborn screening positive predictive value for some conditions (short chain, 3-methylcrotonyl-CoA carboxylase) is low, and many variants are benign, so confirmatory tests must be used before dietary restrictions.
• Results do not give residual enzyme activity, severity or prognosis, so genotyping, enzyme assay or fibroblast flux studies are used.
• Access is limited in low resource settings, where diagnosis relies on clinical suspicion, glucose, ketone and lactate measurements, and samples sent to reference laboratories.

Special groups.

• Neonates and infants: highest risk of decompensation; sampling at 24 to 72 hours of feeding with repeat samples when borderline.
• Pregnant women: ask about acute fatty liver of pregnancy or HELLP when an affected foetus carries a long chain defect; maternal carnitine transporter defects are found through a newborn's low free carnitine.
• Adults: myopathic presentations (recurrent rhabdomyolysis after exercise, fasting or infection) from carnitine palmitoyltransferase 2 deficiency and long chain defects.
• Patients on valproate, dialysis or parenteral nutrition: interpret low carnitine in context.
• Regions: prevalence of medium chain acyl-CoA dehydrogenase deficiency is low in African and Asian populations, but other defects occur, and consanguinity raises the frequency of recessive disorders.

Follow up tests. Urine organic acids, plasma amino acids, free and total carnitine, glucose, ketones, ammonia, lactate, blood gas, creatine kinase, liver tests and uric acid at the time of illness, gene sequencing (ACADM, ACADVL, HADHA, HADHB, CPT1A, CPT2, SLC22A5, ETFA, ETFB, ETFDH, PCCA, PCCB, MMUT), enzyme assay or fibroblast acylcarnitine flux studies, echocardiography and electrocardiogram, testing of siblings and parents, and genetic counselling.