Cholesterol

 
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Cholesterol is the most commonly occurring steroid. It is an important precursor of cholesterol esters, bile acids and steroid hormones. It is derived from dietary sources and synthesized in vivo from acetyl-CoA in the liver (main site) and other tissues (intestines, adrenal glands and reproductive organs). Measurement of cholesterol can give an indication of hepatic function, gastrointestinal disease, and metabolic disorders.

Physiology

Cholesterol occurs in blood as part of all lipoproteins, but low density (LDL) and high density lipoprotein (HDL) fractions have the highest concentrations. LDLs are formed from very low density lipoproteins (VLDL) by endothelial lipoprotein lipase. They are responsible for transporting cholesterol to peripheral tissues, by binding to LDL receptors on these tissues, e.g. adrenal glands, ovary and testes. HDLs are synthesized in the liver and gastrointestinal tract and transport cholesterol from tissues to the liver (so-called "reverse" cholesterol transport, which is thought to be minimal in dogs due to the lack of some transferase enzymes eg CTEP). Once in the liver, cholesterol can be incorporated into VLDLs, synthesized into bile acids, esterified to long chain fatty acids or excreted into the bile. Bile is the main route of excretion of cholesterol. Note that visible lipemia in a blood sample is usually due to increased triglycerides not due to increased cholesterol. 

Methodology

A variety of automated enzymatic assays are used to quantify the cholesterol concentration in blood. Most assays employed for the determination of cholesterol concentration are colorimetric, while others utilize O2 sensing electrodes for quantifying cholesterol levels. The cholesterol CHOD-PAP method meets the standards for measuring cholesterol concentration in serum or plasma set by the National Institutes of Health (NIH), and is the method used at Cornell University.

Reaction type

Blanked end-point reaction

Procedure used at Cornell University

  • Cholesterol CHOD-PAP method: In the first step of this three stage reaction, the enzyme cholesterol esterase hydrolyzes cholesterol esters to yield free fatty acids and cholesterol. In the next step, cholesterol oxidase catalyzes the oxidation of cholesterol to cholest-4-en-3-one. Under the catalytic action of peroxidase, hydrogen peroxide produced in the previous reaction oxidizes the chromophore 4-aminophenazone, in the presence of phenol, to the red dye compound 4-(p-benzoquinone-monoimino)-phenazone. The color intensity change at 500-550 nm is measured photometrically and  is directly proportional to the concentration of cholesterol in the sample. The results are reported out as mg/dL.
  • Reactions are shown below:

cholesterol ester + H2 cholesterol esterase > cholesterol + fatty acids

cholesterol + O2  cholesterol oxidase cholest-4-en-3-one + H2O2

2H2O2 + 4-aminophenazone + phenol  peroxidase > colored complex + 4H2O

 

Units of measurement

Cholesterol concentration in serum is measured in mg/dL (conventional units) or mmol/L (SI units). The conversion formula is shown below:

mg/dL x 0.0259 = mmol/L mmol/L x 38.91 = mg/dL

Reference intervals for cholesterol concentration in domestic species have been established by the Clinical Pathology Laboratory in the Animal Health Diagnostic Center at Cornell University.

Sample considerations

Anticoagulant

Heparin or EDTA

Sample type

Serum or plasma. Dogs and cats should be fasted for around 12 hours to avoid cholesterol interference from post-prandial hyperlipidemia.

Stability

The reported stability of total cholesterol is 5 - 7 days at 4°C, 3 months at -20°C and can last years if stored at -70°C.

Interferences

  • Lipemia: No known interference until >1250 (samples are usually ultracentrifuged to clear as much chylomicrons as possible).
  • Hemolysis: May falsely increase concentrations (hemolysis index > 700 units).
  • Icterus: May falsely decrease concentrations (icteric index > 10).

Test interpretation

Increased concentration (hypercholesterolemia)

High cholesterol is usually due to increased numbers of cholesterol-rich lipoproteins, i.e. HDL and LDL. Because VLDL do contain some cholesterol (12%), high cholesterol can also be seen with very high VLDL concentrations. Chylomicrons have very little cholesterol, so high cholesterol concentrations are not usually seen post-prandially. Common causes of high cholesterol without triglycerides in dogs are nephrotic syndrome, hypothyroidism and cholestasis. Increases of cholesterol and triglycerides in dogs are seen in metabolic conditions such as diabetes mellitus, hyperadrenocorticism, pancreatitis etc (due to high VLDL). High cholesterol in cats is usually due to cholestasis (not all cholestatic dogs and cats will have high cholesterol). Cholesterol is not routinely measured in large animals, therefore we know less about cholesterol concentrations in these species.
  • Artifact: Severe hemolysis may increase values.
  • Physiologic: Mild increases in cholesterol may be seen, although concentrations are not usually increased above the upper reference limit.
  • Pathophysiologic:
    • Nephrotic syndrome: This is characterized by edema, hypoalbuminemia, hypercholesterolemia and proteinuria, due to albuminuria with high urine protein to creatinine ratios (although not all may be present in one animal) and is caused by glomerular damage, e.g. amyloidosis, immune-complex glomerulonephritis. In humans this is mostly due to increased LDL. The exact mechanism is unknown but the following have been postulated: increased VLDL production due to hypoalbuminemia or decreased oncotic pressure (there is no real support for this), defective LDL/HDL processing (loss of plasma constituents in the urine that permit removal of LDL/HDL from circulation could be contributing to this defective processing), increased production of cholesterol-rich lipoproteins or defective conversion of cholesterol to bile acids.
    • Hypothyroidism: In dogs, hypothyroidism is associated with mild to marked elevations in cholesterol, due to increased LDL and HDL. A cholesterol concentration > 750 mg/dL is associated with a risk of atherosclerosis. The cause of the increase is multifactorial and may be partly due to a down-regulation of LDL-receptors in the liver. Thyroid hormone (T3) stimulates LDL receptors (and promotes uptake of cholesterol), therefore lack of thyroid hormone results in decreased LDL receptors and decreased LDL (cholesterol) uptake.
    • Cholestasis: Cholesterol is normally excreted in bile. Cholestasis can result in production of a cholesterol-rich lipoprotein called lipoprotein-X, but the reasons why and how this lipoprotein is formed is unclear. Studies in cats suggest that increased cholesterol is due to extrahepatic and not intrahepatic cholestasis.
    • Other metabolic conditions
      • Diabetes mellitus: Insulin stimulates lipoprotein lipase, which is responsible for hydrolysis of chylomicrons (CM) and VLDL. Insulin also antagonizes hormone-sensitive lipase, the hormone responsible for lipolysis of adipose tissue. Insulin lack results in increased concentrations of CM and VLDL in the blood, with high triglyceride and cholesterol concentrations (although CM and VLDL consist mostly of triglycerides, they also contain small amounts of cholesterol). Lack of inhibition of hormone-sensitive lipase causes increased lipolysis, with increased non-esterified fatty acid presentation to the liver and VLDL production. In addition, LDL receptors on hepatocytes are downregulated, resulting in increased LDL levels.
      • HyperadrenocorticismHypercholesterolemia is due to increased LDL, thought to be due to peripheral insulin resistance and down-regulation of LDL receptors in the liver. Corticosteroids also stimulate hormone-sensitive lipase, resulting in increased lipolysis and VLDL production.
      • Pancreatitis: Although hypertriglyceridemias are more common in this disorder, high cholesterol may be seen concurrently due to inhibition of lipoprotein lipase.
      • Excessive negative energy balance: In states of excessive negative energy balance (e.g. starvation, anorexia) particularly when energy demands are high (e.g. late pregnancy, early lactation), lipolysis of fat stores in adipocytes will increase VLDL concentrations. Although VLDLs contain more triglycerides than cholesterol, increases in both of these substances may be seen. Hyperlipemia due to excessive negative energy balance mostly occurs in horses and camelids and is associated with secondary hepatic lipidosis. In contrast, ruminants with excessive negative energy balance rarely develop triglyceride or cholesterol abnormalities (which has been attributed to inefficient export of VLDL by the liver in these species).
    • Inherited disorders of lipid metabolism: Familial hypercholesterolemia has been reported in Briards, Rottweilers, Shetland Sheepdogs, and Dobermans. Although cholesterol is moderately to markedly increased in these conditions, proportionally lower (milder) increases in triglycerides also occur. Other inherited lipid disorders, e.g. hyperlipidemia of Miniature Schnauzers, hyperchylomicronemia of cats, usually result in increased triglycerides primarily, but you may also see increased cholesterol.

Decreased concentration (hypocholesterolemia)

Low cholesterol can be due to decreased numbers of cholesterol-containing lipoproteins (LDL, HDL, VLDL) or a decreased cholesterol content of these lipoproteins. The most common causes of low cholesterol are protein-losing enteropathy in dogs and cancer in dogs and cats.
  • Artifact: Severe icterus may decrease concentrations.
  • Pathophysiologic
    • Genetic defect in apoprotein production: A genetic defect in the ApoB gene has been identified as a cause of severe hypocholesterolemia and hypotrigyceridemia in Holstein calves. Calves present with ill-thrift and chronic diarrhea from fat malabsorption. They are typically homozogyous for the mutation, which has been traced back to one founder sire (Menzi et al 2016Mock et al 2016).
    • Decreased absorption: Malabsorption and maldigestion problems, e.g. protein-losing enteropathies, exocrine pancreatic insufficiency. A combination of low albumin, low globulins (normal A:G ratio) and low cholesterol is classic for protein-losing enteropathies, whereas a high cholesterol may be seen with protein-losing nephropathy (see above).
    • Decreased production: Since the liver is the main site of cholesterol production, low cholesterol values can be seen in chronic liver diseases (e.g. cirrhosis), synthetic liver failure (acute or chronic), and portosystemic shunts (acquired or congenital). Inflammatory cytokines (e.g. IL-1, IL-6, TNFα) have been shown to decrease hepatic synthesis and secretion of lipoproteins. Low cholesterol is a feature of cats with multiple myeloma (and has been attributed to decreased cholesterol production due to increased oncotic pressure from the paraprotein or high monoclonal protein) (Patel et al., 2005). This could potentially be a mechanism for low cholesterol in dogs with histiocytic sarcoma, particularly the hemophagocytic variant (low albumin and cholesterol are a feature of this tumor in many dogs) (Moore et al., 2006).
    • Altered metabolism: Inflammatory cytokines can reduce the cholesterol content of lipoproteins by decreasing lecithin-cholesterol acyltransferase activity (the enzyme responsible for converting free cholesterol to cholesterol ester which is then incorporated into HDL and LDL). Similarly, inflammatory cytokines can reduce lipoprotein lipase activity (the enzyme facilitates the conversion of VLDL to LDL). This would lower cholesterol through decreased lipoprotein number and cholesterol content.
    • Increased uptake of lipoproteins: Upregulation of LDL-receptors on cells (peripheral tissues and liver) can potentially lower cholesterol values. This occurs in rapidly proliferating tumor cells (e.g. acute myeloid leukemia in human patients) and in response to inflammatory cytokines (some acute phase proteins in human patients, such as serum amyloid A, enhance LDL removal from the circulation in acute phase reactions). 

Related links

  • Clinical Pathology Laboratory in the Animal Health Diagnostic Laboratory at Cornell University website: Information on cholesterol testing. This is part of our routine small, but not large, animal panels.
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