Biochemistry, Ketone Metabolism

Molecular Level

A ketone is a name for a specific elemental structure in organic chemistry. A ketone consists of a single bond to 2 CH3 or R groups with a double bond to an oxygen molecule. Acetone, 3-B-hydroxybutyrate (3HB), and acetoacetate all contain a ketone group and are therefore very soluble in the body tissues. The solubility of these ketones allows them to be transported throughout the body to various tissues. The important rate-limiting steps in ketone metabolism include hormone-sensitive lipase (HSL), acetyl-CoA carboxylase, succinyl-CoA-oxoacid transferase (SCOT), and HMG-CoA synthase. HSL and HMG CoA synthase are inhibited by insulin and stimulated by glucagon. Acetyl-CoA carboxylase is stimulated by insulin and inhibited by glucagon. All 3 of these enzymes have the same effect: slowing ketone production in the presence of insulin and increasing it in the presence of glucagon. Finally, increased levels of acetoacetate in the mitochondria of target organs inhibit SCOT, thereby reducing ketone metabolism.[2][3][4]

Mechanism

The process of ketogenesis begins with fatty acyl-CoA molecules. These molecules arise from the lipolysis of long-chain fatty acids via hormone-sensitive lipase. Triglycerols and amino acids may also be sources for Acetyl CoA; however, these sources usually add up to less than 10% of the total. The regulation of hormone-sensitive lipase (HSL) is via negative feedback from increases in insulin and glucose concentration. Positive feedback from glucagon and beta-adrenergic catecholamines increases HSL activity to provide more fatty acyl-CoA molecules. HSL regulation takes place via phosphorylation by protein kinase A (PKA). PKA is activated by cyclic AMP (cAMP), which is directly downstream from the cell surface receptor affected by hormones. Fatty acids pass through the cell membrane and circulate in the blood. Certain tissues of the body, such as skeletal muscle, myocardium, and liver, can use fatty acids as an energy source, whereas the brain cannot use fatty acids for energy and must use ketone bodies as a means of transporting energy from fat stores.

Fatty acids in the blood are converted to ketone bodies when insulin is low, and the fatty acid concentration is high. Fatty acyl-CoA is transported into the liver mitochondria by the carnitine shuttle system. This system involves 2 transmembrane proteins to move fatty acyl-CoA molecules across the mitochondrial membrane. The first protein is carnitine palmityl transferase I (CPT I), which, on the cytosolic side of the mitochondrial membrane, transfers the fatty acyl-CoA across the outer membrane. During this process, a carnitine molecule is attached to the fatty acyl-CoA molecule to make an acylcarnitine. The acylcarnitine is carried through the mitochondrial matrix by a transporter protein called carnitine/acylcarnitine translocase. At the inner mitochondrial membrane, the acylcarnitine molecule is converted back to acyl-CoA and carnitine by CPT 2.

Ketone synthesis in the liver produces acetoacetate and beta-hydroxybutyrate from 2 acetyl-CoA molecules. This process begins in the liver mitochondria after the fatty acyl-CoA molecule is transported across the inner mitochondrial membrane via the carnitine shuttle. The fatty acyl-CoA molecules undergo beta-oxidation to become acetyl-CoA molecules. Acetyl CoA molecules are either converted to malonyl CoA by acetyl CoA carboxylase or acetoacetyl CoA by 3-ketothiolase. Malonyl-CoA serves as a negative feedback on liver CPT-1. Acetoacetyl CoA is further converted to 3-hydroxy-3-methylglutaryl CoA (HMG CoA) by HMG CoA synthase. HMG CoA synthase is essential to this process, as it is the rate-limiting step for the synthesis of ketone bodies. HMG CoA synthase regulation is influenced positively by glucagon and negatively regulated by insulin. HMG CoA is finally converted to acetoacetate by HMG CoA lyase. At this point, acetoacetate may be converted to 3-B-hydroxybutyrate (3HB) by 3HB dehydrogenase. Acetoacetate and 3HB are organic acids that diffuse freely across cell membranes into the blood and other organs of the body.

Upon arrival in the mitochondria of distant organs, ketone bodies are utilized for energy. The first step involves an enzyme that converts acetoacetate to acetoacetyl CoA. The enzyme responsible for this conversion is called succinyl-CoA-oxoacid transferase (SCOT), and it is the rate-limiting step in the utilization of ketones for energy. High concentrations of acetoacetate feed back negatively on SCOT, decreasing ketone conversion. Finally, acetoacetyl CoA is converted to acetyl CoA by methylacetoacetyl CoA thiolase. Acetyl-CoA can be converted to citrate and cycled through the citric acid cycle to produce FADH2 and NADH, or converted to oxaloacetate and used in gluconeogenesis.[2][3][4][5][6]

Testing

A traditional method to detect elevated ketones is to smell the patient's breath. Acetone and other ketone bodies have a distinct fruity odor detectable by a clinician. A urine dipstick test can be used to qualitatively detect the presence of ketone bodies. A nitroprusside stain detects acetone and acetoacetate in the urine. The urine test uses a scale of 0 to +4, where 0 is undetectable, and +4 indicates a high amount. New quantitative lab studies have recently become available that measure 3HB in serum.[5][7]

Clinical Significance

The most common clinical manifestation associated with ketone bodies is ketoacidosis. Ketosis is usually a normal physiological state. However, if unregulated, it may also drive life-threatening syndromes termed ketoacidosis. The most common forms of ketoacidosis are diabetic ketoacidosis (DKA) and alcoholic ketoacidosis. DKA most commonly results from an inadequate response to insulin. This lack of insulin response could be due to noncompliance with treatment, undiagnosed type 1 DM, or subtherapeutic insulin administration. A lack of insulin response results in increased glucagon and decreased glucose uptake. Decreased insulin also facilitates the increased activity of hormone-sensitive lipase. Hormone-sensitive lipase converts triglycerides to fatty acyl-CoA molecules. Fatty acyl-CoA molecules overload the Krebs cycle and shunt over to ketone metabolism. An abundance of ketone bodies causes an anion gap acidosis. Patients also usually present with hyperglycemia, lethargy, abdominal pain, polyuria, polydipsia, vomiting, and mental status changes.

Alcoholic ketoacidosis occurs in chronic alcoholics after an abrupt withdrawal or acute intoxication. The ethanol is metabolized to acetic acid, and NAD+ is converted to NADH while acting as a coenzyme. Acetic acid is shunted to ketogenesis if insulin and glucagon are at favorable concentrations, such as in hypoglycemia. Additionally, NADH inhibits gluconeogenesis because NAD+ is required for multiple steps in gluconeogenesis. Finally, during the acute withdrawal of ethanol, the release of epinephrine further drives ketogenesis and, at that point, ketoacidosis. Some inherited defects in ketone metabolism also exist. The most common inherited deficiencies include a systemic primary carnitine deficiency and medium-chain acyl-CoA dehydrogenase deficiency.

A patient with a systemic primary carnitine deficiency (CDSP) is unable to transport fatty acyl-CoA molecules into the mitochondria in the liver. CDSP has an autosomal recessive inheritance pattern and is caused by mutations in the SLC22A5 gene on chromosome 5. Patients can present as infants with hyperammonemia, or they may present as adults with elevated liver enzymes and hypoketotic, hypoglycemic episodes. Regardless of the onset, the disease may be diagnosed by genetic testing or by measuring blood carnitine levels. Treatment of these patients includes supplementing with carnitine, reducing dietary fat, and shortening fasting durations.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency occurs in about 1 in 10,000 White infants. The MCAD deficiency screen is a common test included in a standard newborn screening. MCAD is an enzyme that converts 4-12 carbon fatty acyl-CoA molecules into acetyl-CoA in the mitochondria for utilization. MCAD has an autosomal recessive inheritance pattern, most commonly due to mutations in the ACADM gene on chromosome 1. These patients may experience seizures, comas, hypoketotic hypoglycemia, and failure to thrive if not diagnosed early. The mainstay of treatment for these patients is early diagnosis and avoidance of fasting.[7][8][9][10]