Cholesterol screening can be performed with or without fasting and should include total and HDL cholesterol tests. The frequency of cholesterol testing depends on the patient's risk for CAD. Adults over 20 with total cholesterol levels below 200 mg/dL need to be tested once every five years. People with higher levels should be tested for LDL cholesterol and tested at least once per year thereafter, if the LDL cholesterol is 130 mg/dL or higher. The National Cholesterol Education Program (NCEP) suggests further evaluation when the patient has any of the symptoms of CAD or if she or he has two or more of the following risk factors for CAD:
Measurements of cholesterol and triglycerides are routinely performed using enzymatic methods. For cholesterol, the cholesterol oxidase method is used. Plasma or serum is mixed with a reagent containing cholesterol ester hydrolyase, cholesterol oxidase, peroxidase, and a chromogen. The cholesterol ester hydrolyase converts cholesterol esters (cholesterol coupled to a fatty acid) to free cholesterol. This reacts with cholesterol oxidase forming an oxidation product and hydrogen peroxide. The peroxidase enzyme catalyzes the oxidation of the chromogen by the hydrogen peroxide. This forms a red colored product that can be measured with a spectrophotometer. The amount of light absorbed at 500 nm is directly proportional to cholesterol concentration. HDL cholesterol is usually measured by the same reaction except that the enzymes are coupled to polyethylene glycol (PEG). In the presence of sulfated cyclodextrin, these enzymes will not react with the cholesterol in LDL, VLDL, or chylomicrons. LDL cholesterol is measured by first precipitating the other lipoproteins using a mixture of antibodies to apolipoprotein C and apolipoprotein E. The LDL cholesterol can be separated by centrifugation and then measured using the cholesterol oxidase reaction. Alternatively, LDL cholesterol can be calculated using the Friedewald formula. LDL cholesterol = total cholesterol minus (HDL cholesterol + triglyceride/5). This formula will underestimate LDL cholesterol when triglycerides are above 400 mg/dL.
Triglycerides are routinely measured using the glycerol kinase reaction. The reagent contains the enzymes lipase, glycerol kinase, glycerol phosphate oxidase, and peroxidase. It also contains adenosine triphosphate (ATP) and a chromogen. Triglycerides are composed of glycerol that is bound (esterified) to three long chain fatty acids. The lipase sequentially splits the fatty acids from the molecule forming glycerol and free fatty acids. The glycerol kinase catalyzes the transfer of phosphorus from ATP to the glycerol forming glycerol-phosphate. The glycerol phosphate oxidase is used to oxidize this to dihydroxyacetone phosphate. This reaction generates hydrogen peroxide. In the final step, the peroxidase enzyme catalyzes the oxidation of the chromogen by the hydrogen peroxide. This forms a red-colored product that can be measured with a spectrophotometer. The amount of light absorbed at 500 nm is directly proportional to triglyceride concentration. An important potential interfering substance in this reaction is glycerol, which is a common additive to many medications. If the Friedewald formula is used to calculate LDL cholesterol, the triglyceride measurement must be corrected by subtracting the plasma glycerol concentration from the triglyceride result.
Jane E. Phillips, The Gale Group Inc., Gale, Detroit,