Cholesterol

Cholesterol is the most common steroid in the body. 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). 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

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.
  • Reactions are shown below:

cholesterol ester + H₂O  ^{cholesterol esterase} > cholesterol + fatty acids

cholesterol + O₂  ^{cholesterol oxidase} > cholest-4-en-3-one + H₂O₂

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

• Lipoprotein quantification: At Cornell University, lipoprotein classes can be subfractionated using polyacrylamide gel electrophoresis (Lipoprint, Quantimetrix) (Behling-Kelly et al 2022).

 

Units of measurement

Cholesterol concentration in serum or plasma 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

Sample considerations

Anticoagulant

Heparin or EDTA for total cholesterol

Sample type

Serum or plasma. Dogs and cats should be fasted for around 12 hours.

Stability

The reported stability of total cholesterol is 7 days at 4°C, 3 months at -15 to -20°C (per product information sheet) and can last years if stored at -70°C.

Interferences

• Lipemia: No known interference up to 2000 units (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 > 15 or 14 mg/dL for unconjugated bilirubin and 16 mg/dL for conjugated bilirubin (per product sheet).

Test interpretation

Increased concentration (hypercholesterolemia)

High total 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 (however, 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: A study in healthy dogs showed that cholesterol concentrations did not increase beyond the total allowable error for the analyzer after feeding (Yi et al 2022).
• Iatrogenic: Exogenous corticosteroids can result in a fasting hypercholesterolemia in dogs and cats (Lowe et al 2008, Khelik et al 2019, Tinklenberg et al 2020), although there are far higher fold increases in triglycerides than cholesterol.
• 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. High cholesterol concentrations occur in dogs and cats with extrahepatic bile duct obstruction and cats with experimental bile duct obstruction (Center et al 1983, Center 2009), although is more frequently seen in dogs with naturally occurring bile duct obstruction (Center 2009). Cholangiohepatitis can also result in high cholesterol concentrations, likely due to cholestasis associated with biliary involvement or obstruction (Center 2009, Brown et al 2000), however cholesterol concentrations are not invariably increased and are not clearly associated with high total bilirubin concentrations (Fuentealba et al 1997). High cholesterol concentrations are not typically seen in cats with hepatic lipidosis, for unclear reasons (possible downregulation of production due to lipid-induced hepatic dysfunction) (Brown et al 2000, Minamoto et al 2019). It is unclear if functional cholestasis induces hypercholesterolemia in dogs or cats. The situation is less clear in large animals, because cholesterol is not routinely measured on chemistry panels. No increases in cholesterol concentration were seen in ponies with experimentally induced bile duct obstruction, despite biochemical evidence of cholestasis (increased conjugated or direct bilirubin concentrations) (Bauer et al 1990)
  • 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. In one study in cats, cholesterol concentrations were increased in 80% of cats, some but not all of which had increased total bilirubin concentrations (Zini et al 2010). 
    • Hyperadrenocorticism: Hypercholesterolemia 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 homozygous for the mutation, which has been traced back to one founder sire (Menzi et al 2016, Mock 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 or alter their lipid composition in humans. Low cholesterol is a feature of cats with multiple myeloma and has been postulated to be due to decreased production from increased oncotic pressure from the paraprotein or high monoclonal immunoglobulin (Patel et al 2005). However, there is no direct link between hepatic receptors that sense increased pressure in the sinusoids (baroreceptors) and cholesterol metabolism, so other mechanisms could be operative, including increased uptake by tumors (see below). Dairy cattle with lipidosis can have low cholesterol and an increased non-esterified fatty acid (NEFA) to cholesterol ratio (>0.2 in SI units) may be indicative of lipidosis and more sensitive than other liver markers, such as AST activity (Mostafavi et al 2013).
  • 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. Dogs with septic and non-septic inflammation have a trend towards lower cholesterol concentrations than healthy dogs, with alterations in the lipoprotein fractions with the Lipoprint assay (decrease in a subfraction of HDL and an increase in LDL, particularly in septic dogs). The mechanism behind these changes is unknown (Behling-Kelly et al 2022).
  • Increased uptake of lipoproteins: Upregulation of LDL-receptors on cells (peripheral tissues and liver), such as SBR-1, can potentially lower cholesterol concentrations. This occurs in rapidly proliferating tumor cells and has been attributed to inflammatory cytokines in people (some acute phase proteins in human patients, such as serum amyloid A, enhance LDL removal from the circulation in acute phase reactions). We suspect that increased uptake or use of cholesterol is the main reason for low cholesterol concentrations seen in some animals with cancer, including dogs with acute myeloid leukemia and histiocytic sarcoma, particularly the hemophagocytic variant (low albumin and cholesterol are a feature of this tumor in some dogs) (Moore et al., 2006), and cats with multiple myeloma (Patel et al 2005).
  • Unknown mechanism: Dogs with Addison’s disease (hypoadrenocorticism) frequently have low cholesterol concentrations. The mechanism is unclear (Lyngby and Sellon 2016).