Recommendation for Researchers
Total Cholesterol and HDL
At a minimum, Total Cholesterol and HDL should be measured in all studies that evaluate cardiovascular risk (cost to MDRC members = $10.00 including both assays). These measures do not require a fasting serum specimen.
Because of the high rate of hypertriglyceridemia in T2D, direct LDL measures should be strongly considered as the preferred LDL assay, as it does not have to be performed on a fasting sample (cost to MDRC members = $12.00). Therefore, the only commonly used cholesterol measurement that still requires a fasting specimen is the TG measurement. If you do not have a direct LDL-C measure available and the TG level is >250-400 mg/dl, we recommend one of two options. If the TG is between 250 and 400, the calculation of LDL-C is less accurate, but could still be used to calculate the LDL-C, however, when the TG level is greater than 400mg/dl, the accuracy of the calculation further deteriorates, and by convention, is usually not recommended. However, the Total Non-HDL Cholesterol level (TNH-C = Total Cholesterol–HDL-C) is the single greatest predictor of cardiovascular risk and can be used as a surrogate measure of lowering of cardiovascular risk. The main problem with this approach is that most clinicians are unfamiliar with this measure.
Even if HDL-C is measured with a direct assay that is relatively unaffected by high lipid concentrations, it is not recommended to use the HDL-C values to calculate LDL-C (see Appendix) if TG levels are in excess of 400 mg/dl. The new NCEP guidelines set the optimal HDL-C level as being >40mg/dl.
- A. Rifal N, Iannotti E, DeAngelis K, Law T. Analytical and clinical performance of a homogeneous enzymatic LDL-cholesterol assay compared with the ultracentrifugation-dextran sulfate-Mg2+ method. Clinical Chemistry 44: 1242-1250 (1998).
- B. Benlian P, Cansier C, Hennache G, Khallouf O, Bayer P, Duron F, Carrat F, Couderc R, Chazouilleres O, Bardet J, Bouchard P, Poupon R, Masliah J, Bereziat G. Comparison of a new method for the direct and simultaneous assessment of LDL- and HDL-Cholesterol with ultracentrifugation and established methods. Clinical Chemistry 46:493-505 (2000).
- C. Kwiterovich Jr. P.O. The metabolic pathways of high-density lipoprotein, low-density lipoprotein and triglycerides: A current review. Am J Cardiol 86 (suppl): 5L-10L (2000).
Measurement Methods for Specific Types of Lipoprotein Cholesterol
The major forms of lipoprotein cholesterol routinely measured for clinical evaluations are high-density lipoprotein cholesterol (HDL-cholesterol) and low-density lipoprotein cholesterol (LDL-cholesterol). The approaches in both cases involve the selective exposure of the relevant type of cholesterol to the esterase/oxidase/chromogen reactions. This strategy involves the removal of the undesired or “irrelevant” form of cholesterol from the final, color-forming reaction. For instance, if the purpose of the assay is to measure LDL-cholesterol, it is possible to precipitate or eliminate the non-LDL forms of cholesterol (e.g., HDL-cholesterol) and make them inaccessible to the final color forming reaction which would only measure the cholesterol present in LDL.
The strategies used to select the form of cholesterol that is relevant to the evaluation of the patient involve the following overall approaches:
This method allows for the selection of specific types of lipoproteins based on their density following ultracentrifugation and the isolation of the relevant centrifugate fraction for subsequent measurement of the cholesterol present. This method follows closely the definition of lipoproteins based on their density and is the core of the Centers for Disease Control reference method used to standardize other analytical approaches. The advantages are its conceptual accuracy and the ability to physically select the relevant form of cholesterol for subsequent measurement. The disadvantages of ultracentrifugation methods are that the procedure is time and labor consuming, difficult to automate and requires investment in equipment (e.g., ultracentrifuges) that many laboratories may not find easy to afford.
Sequestration or Blocking Assays
These methods are easier to automate and can be manufactured commercially as kits for autoanalyzers. The classical example is the use of antibodies or other compounds to precipitate or block the forms of lipoporotein cholesterol that are not of interest and make them inaccessible for the subsequent enzymatic reactions. The success of this approach largely depends on the efficacy of the sequestering or blocking compounds to act on the “unwanted” lipoproteins.
These assays also are easy to automate and are commercially available as kits for autoanalyzers. The approach in this case is the use of specific buffers and surfactants to preferentially expose the “unwanted forms” of cholesterol to the action of cholesterol esterase and cholesterol oxidase in a non-color-forming reaction, thus eliminating the “unwanted or irrelevant” forms of cholesterol from the reaction mix. This elimination step is followed by the subsequent addition of specific buffers to expose the “relevant” form of cholesterol to the action of cholesterol esterase and cholesterol oxidase in a final color-forming reaction (i.e., coupled with a chromogen), that can be used to measure the concentrations of relevant cholesterol spectrophotometrically.
Types of Lipid Assays at the Chemistry Laboratory
The Chemistry Laboratory measures triglycerides with an enzymatic colorimetric kit by Roche for the Cobas Mira Chemstation. The triglycerides in the sample are hydrolyzed by lipoprotein lipase to glycerol and fatty acids. The glycerol is then phosphorylated to glycerol-3-phosphate by glycerol kinase and subsequently catalyzed by glycerol oxidase to form dihydroxyacetone phosphate and hydrogen peroxide. The hydrogen peroxide is then reacted in the presence of peroxidase to form a chromogen whose increases in absorbance are proportional to the concentration of triglycerides.
Total Cholesterol Determination
Total cholesterol is measured at the Chemistry Laboratory with an enzymatic kit manufactured by Roche for the Cobas Mira Chemstation. This method follows a two-step approach. In the first step, cholesterol is desterified by the action of cholesterol esterase, and subsequently, it is exposed to the action of cholesterol oxidase. This second step is in turn coupled to a chromogen (color-forming compound) which can be measured with the aid of a spectrophotometer. The increases in absorbance given by the chromogen are proportional to the concentrations of cholesterol in the sample.
This esterase/oxidase/chromogen approach is the basis of many of the commercially available kits for the measurement of total cholesterol. It also is a central part of the strategies for the measurement of the cholesterol present in the different lipoprotein fractions in blood.
In the Chemistry Laboratory, HDL Cholesterol is measured with an HDL direct kit for the Cobas Mira Chemstation. This kit has been reported to meet the NCEP guidelines for precision and accuracy and its calibrators are traceable to the CDC reference method. This kit follows an elimination approach in which surfactants are used to promote the elimination of cholesterol in non-HDL lipoproteins in a primary non-color forming reaction. This step is followed by the addition of a second reaction mix in which HDL-cholesterol is selectively exposed to the action of cholesterol esterase and cholesterol oxidase in a color forming reaction that is measured spectrophotometrically.
This kit is reported to be relatively unaffected by high lipid concentrations and would therefore be less sensitive to hypertriglyceridemia. However, converting HDL to LDL values using the Friedewald equation when triglyceride concentrations are above 400 mg/dl is not recommended.
HDL cholesterol has frequently been used to calculate LDL-cholesterol, utilizing the Friedewald equation:
[LDL-Cholesterol = Total Cholesterol – (HDL-Cholesterol + Triglycerides/5)]
(All concentrations as mg/dl)
The problem with this approach is that because it involves measurements other than LDL cholesterol, it is subject to potential compounded inaccuracies from the determinations of the other lipids in the equation. Another very important shortcoming is its limited usefulness when assaying blood samples when triglyceride levels are above 400 mg/dl.
MDRC's Chemistry Laboratory performs the direct measurement of LDL cholesterol with a homogenous, elimination LDL-Cholesterol kit for the Cobas Mira Chemstation. The method uses a primary detergent to first solubilize non-LDL lipoprotein particles. The cholesterol from these fractions is released and consumed in a non-color-forming reaction. Following this step, a second detergent specific for the solubilization of the LDL fraction is added in the presence of a chromogenic coupler for the detection of LDL-cholesterol. The enzyme reaction, in the presence of the coupler, produces color that is proportional to the concentration of LDL-cholesterol. This assay has the advantage of measuring LDL-cholesterol directly and of not being affected by high triglyceride values of up to 1200 mg/dl. The assay meets the National Cholesterol Education Program guidelines for accuracy, precision and total error, and its calibrators are traceable to the CDC reference method.
NCEP Criteria for Standardization of Cholesterol Assays
In order to promote the standardization of cholesterol measurements, the National Cholesterol Education Program has issued performance guidelines for the measurement of HDL-cholesterol and LDL-cholesterol. It is important that the laboratory conducting the determinations utilizes a type of assay or kit that meets the NCEP guidelines for precision and accuracy. In addition to these guidelines, it is important to ensure that the assay in question uses calibrators that are traceable to the CDC reference method. To further promote the standardization of cholesterol measurements, CDC has created the Cholesterol Reference Method Laboratory Network to assist commercial manufacturers and specific laboratories with the validation of their assays as well as with the traceability of the determinations to the CDC reference method. Their services include accurate reference methods, reference materials and verification sets of sera for the standardization of assays.