Free and Total Carnitine

Carnitine is a quaternary, water-soluble ammonia compound biosynthesized from lysine and arginine. It serves as a mechanism for transport of long-chain fatty acids from the cytoplasm across the inner mitochondrial membrane and into the mitochondrial matrix, the site of b-oxidation of fatty acids for energy generation.

The reference range of carnitine depends on the laboratory being used; various ranges have been reported in the literature. ^[1]

The following table of values is derived from Belay et al 2006. ^[2] These examples do not imply endorsement of the laboratories cited, but instead are used for reference only.

Table. Reference Range of Carnitine at a Single Laboratory ^[2] (Open Table in a new window)

Quest Laboratories reports the reference range of total carnitine as follows ^[3] :

• Men: 30-70 μmol/L

• Women: 25-58 μmol/L

• Male children (age ≤17 years): 32-62 μmol/L

• Female children (age ≤17 years): 28-59 μmol/L

Quest Laboratories reports the reference range of free carnitine as follows ^[3] :

• Men: 23-59 μmol/L

• Women: 19-48 μmol/L

• Male children (age ≤17 years): 25-54 μmol/L

• Female children (age ≤17 years): 19-51 μmol/L

The University of California San Francisco Laboratory reports the normal values of free and total carnitine in adults as 18-69 μmol/L and 20-71 μmol/L, respectively. ^[4]

Chace et al (2003) examined free and total carnitine levels in newborns. The reference ranges depended both on technique used (radioenzyme vs tandem mass spectroscopy) and the sample type (whole blood vs serum). ^[5]

Variations in the ratio of free to total carnitine may also be important; typically, normal is reported as 0.1-0.4. ^{[2, 6, 7]}

Carnitine is an important, small water-soluble molecule that binds to long-chain fatty acids and facilitates their transport across the inner mitochondrial membrane and into the mitochondrial matrix to undergo fatty acid oxidation (metabolism).

Carnitine is derived from both the diet (meats and milk) and synthesis (very slowly) from trimethyllysine. However, most carnitine in the body is intracellular; it is not metabolized, and excretion in the urine is very limited owing to very efficient reuptake by the of organic cation transporter receptor type 2 (OCTN2) transporter (see below). ^{[6, 7, 8]}

Free and total carnitine levels within the reference range typically indicate adequate intake, stores, and metabolism. Some disease states may show abnormal carnitine levels only during a stressed state.

Causes of low carnitine levels can be genetic (due to various gene mutations) or acquired (secondary to medication use or a disease state).

Decreased carnitine values

Primary carnitine deficiency is caused by an autosomal-recessive defect in the SLC22A5 gene, resulting in a lack of OCTN2, which is a high-affinity carnitine-uptake transporter expressed in muscle, kidney, and heart. ^[9]

Laboratory values in primary carnitine deficiency show markedly decreased free and total carnitine levels, since 90-95% of filtered carnitine is lost in the urine. Analysis of urine organic acids, serum amino acids, and acylcarnitine panels can be used to distinguish this condition from other causes of carnitine deficiency. ^{[8, 10, 11]}

Individuals with primary carnitine deficiency usually present with cardiomyopathy and skeletal weakness or with episodic hypoketotic hypoglycemia and encephalopathy when stressed at around age 2-4 years. This results from the inability to oxidize fatty acids and generate ketones to provide energy during catabolic states. The disorder is fatal without treatment, but supplementation with oral carnitine results in elevated carnitine levels and prevents progression of the disease. ^{[8, 11, 12, 13]}

A literature review by Crefcoeur et al found that in individuals with primary carnitine deficiency, the most prevalent symptoms were cardiac (23.8% of patients), with cardiomyopathy being the predominant manifestation of these. Neurologic, hepatic, and metabolic symptoms developed in 7.1%, 8.4%, and 9.2% of persons with primary deficiency and occurred most often in early childhood. The condition was asymptomatic in 55.1% of patients with primary deficiency. ^[14]

Carnitine-acylcarnitine translocase deficiency (CACT) typically presents in an autosomal-recessive fashion with seizures, apnea, and an irregular heart beat in the neonatal period (although presentation can occur as late as age 15 months) and results from mutations in the CACT protein (SLC25A20 gene), a carnitine-acylcarnitine exchanger on the inner mitochondrial membrane. Crisis is triggered by fasting, viral illness, or stress (an in other fatty-acid disorders). In addition to low carnitine levels, laboratory studies also show hypoketotic hypoglycemia; elevated levels of ammonia, creatine kinase (CK), liver enzymes, and long-chain acylcarnitines in the blood; and dicarboxylic aciduria in urinary organic acids. CACT is treated with frequent feedings of carbohydrates, medium-chain triglycerides, and carnitine. ^{[7, 11, 15]}

The autosomal-recessive disorder carnitine palmitoyltransferase 2 (CPT-2) deficiency is also characterized by low carnitine levels. The CPT-2 protein is essential for removing carnitine from long-chain fatty acids after translocation into the mitochondrial matrix is and thus essential for fatty acid oxidation. Although it typically presents as a myopathy in adolescents or adults, CPT-2 deficiency can also present as severe fatal neonatal and hepatocardiomuscular infantile forms. The difference in presentation relates to the amount of residual function (genotype-phenotype correlation).

Neonates with CPT-2 deficiency present within days of birth with encephalopathy, cardiomegaly, hepatomegaly, seizures, cardiac arrhythmias, and respiratory distress, and the condition is rapidly fatal. The infantile form presents between ages 6 and 24 months as episodes of encephalopathy, liver failure, seizures, hypoketotic hypoglycemia, metabolic acidosis, elevated CK levels, reversible hepatomegaly, and, in some cases, cardiomyopathy and arrhythmias, precipitated by infection, fasting, or fever.

The adolescent and adult form of CPT-2 deficiency presents with myopathic pain precipitated by exercise, cold, fever, or prolonged fasting and may be associated with myoglobinuria and kidney damage/failure.

Elevated long-chain acylcarnitine levels are detected in all forms of CPT-2 deficiency, and neonatal screening can be useful in determining the cause of death in the neonatal form. ^{[6, 8, 11, 13]}

Secondary carnitine deficiency can result from numerous conditions, such as chronic renal failure, end-stage renal disease, renal Fanconi syndrome, Lowe syndrome, cystinosis, and valproate therapy, all of which cause impaired carnitine reuptake from the kidneys. Carnitine-free diets (such as in those receiving intravenous nutrition), organic acidurias, and urea-cycle defects can also cause deficiency. ^{[2, 6, 11, 16, 17, 18]}

Transient falsely low carnitine levels have been reported in infants born to mothers with primary carnitine deficiency. ^[19]

Elevated carnitine values

Elevated carnitine levels are seen in carnitine palmitoyltransferase 1 (CPT-1) deficiency. The CPT-1 protein conjugates carnitine to long-chain fatty acids, and mutations in the CPT1A gene (expressed in liver cells) present as encephalopathy, seizures, and hypoketotic hypoglycemia in children younger than 18 months, usually triggered by a minor viral illness or fasting.

Blood testing shows low levels of long-chain acylcarnitines (long-chain fatty acid linked to carnitine) and elevated ratios of carnitine: C16+C18. Prevention involves avoidance of fasting (with nighttime feedings of cornstarch) and enrichment of diet with medium-chain triglycerides, which do not require conjugation to enter the mitochondrial matrix for oxidation. ^{[8, 13]}

Normal carnitine values

Numerous defects of fatty acid oxidation and organic acidurias can produce normal or decreased total serum carnitine levels. The reduced carnitine levels result from increased renal losses, since long-chain fatty acids accumulate and compete with carnitine for reuptake in the kidney. Therefore, organic acids and acylcarnitine profiles also show abnormal levels of metabolites.

A full discussion is beyond the scope of this review, but some examples include short-chain acyl CoA dehydrogenase deficiency (SCAD), medium-chain acyl CoA dehydrogenase deficiency (MCAD), very-long-chain/long-chain acyl CoA dehydrogenase deficiency (VLCAD/LCAD), propionic acidemia (PPA), and methylmalonic acidemia (MMA). ^{[11, 13, 20, 21]}

Carnitine levels are highest during a well-fed state (eg, not in a starvation or catabolic state). Repeat studies during a catabolic or fasting state can be useful if initial measurements are within the reference range, although induction of fasting can result in an acute metabolic crisis, leading or morbidity and mortality, and thus should be undertaken with extreme care. Loading tests with carnitine can also be useful, with retesting of carnitine levels to assess response to treatment. ^[13]

Carnitine testing is performed via liquid chromatography/tandem mass spectrometry (LC/MS/MS).

Two mL of blood is collected and sent in a red– or dark-green–top vial (light-green top is not used). The plasma is separated and frozen at -20°C. At least 0.5 mL of serum or heparinized plasma (preferred volume, 1 mL) is sent. ^{[3, 4, 22]}

Some laboratories also offer blood spot testing, and all states require testing for primary carnitine deficiency. ^[23]

If low serum carnitine levels are detected, urine testing may be useful for evaluating for a renal etiology. For urine testing, at least 2 mL of urine needs to be sent, and the sample should be frozen. Shipping should be overnight, optimally with dry ice. Collection requirements can vary with different laboratories, however, and, thus, prior to collection and shipping of specimens, verification by phone with the receiving laboratory should be performed. ^{[3, 4, 22]}

This test can be used in conjunction with acylcarnitine and urine organic acids panels performed as part of the newborn screen or later if carnitine levels are abnormal. Specifically, in different disease states, different metabolites accumulate in the serum or urine, although the panels are more likely to be useful during acute catabolic states. ^{[11, 13]}

Description

Carnitine is a quaternary ammonia water-soluble compound biosynthesized from lysine and arginine. It serves as a mechanism for transport of long-chain fatty acids from the cytoplasm across the inner mitochondrial membrane and into the mitochondrial matrix, the site of b-oxidation of fatty acids for energy generation.

Most carnitine biosynthesis occurs in the liver and kidneys, and vitamin C is required. Meat and dairy products supply dietary carnitine. It is stored in skeletal and cardiac muscle, as well as in the liver and red blood cells. Carnitine is excreted in the urine, and the vast majority of carnitine is reabsorbed in the kidneys. Therefore, secondary carnitine deficiency can result from any condition that decreases carnitine reuptake. ^{[2, 7, 13]}

Indications/Applications

Primary carnitine deficiency is tested for as part of newborn screening tests, although carnitine levels may be normal in a well-fed state. Carnitine levels (free and total) should be obtained, along with acylcarnitine, serum amino acid, and urine organic acid panels, in patients presenting with symptoms suggestive of disorders of fatty acid oxidation. In stressed or catabolic states, affected individuals may decompensate and can present with various symptoms, including encephalopathy, hepatomegaly, hypoketotic hypoglycemic, muscle weakness, apnea, and cardiac arrest, among others. ^{[8, 11, 24, 25]}

As mentioned above, carnitine deficiency can be primary (due to defects in proteins essential for carnitine reuptake or scavenging) or secondary (due to molecules that interfere with reuptake of carnitine from the kidneys). ^[26] It is only by analyzing the acylcarnitine, amino acids, and organic acids profiles that a final diagnosis can be determined.