• Component Description
• Eligible Sample
• Description of Laboratory Methodology
• Analytic Notes
• Codebook
  • SEQN - Respondent sequence number
  • LBXTC - Total cholesterol (mg/dL)
  • LBDTCSI - Total cholesterol (mmol/L)
  • LBDHDL - HDL-cholesterol (mg/dL)
  • LBDHDLSI - HDL-cholesterol (mmol/L)

National Health and Nutrition Examination Survey

1999-2000 Data Documentation, Codebook, and Frequencies

Cholesterol - Total & HDL (Lab13)

Data File: Lab13.xpt

First Published: June 2002

Last Revised: April 2010

Component Description

Total Cholesterol and HDL-Cholesterol

The goals of this component are:

1. to monitor the prevalence and trends in major cardiovascular conditions and risk factors in the U.S.; and
2. to evaluate prevention and treatment programs targeting cardiovascular disease in the U.S.

The main element of the cardiovascular disease laboratory component in NHANES is blood lipid levels. Cardiovascular disease is the leading cause of death in the United States. The data will be used to monitor the status of hyperlipidemia and the success of the National Cholesterol Education Program.

Eligible Sample

Participants aged 3 years and older who do not meet any of the exclusion criteria were sampled.

Description of Laboratory Methodology

 Data Collection Methods

In the mobile examination center (MEC) laboratory, blood specimens are processed, stored, and shipped to the Johns Hopkins University Lipoprotein Analytical Laboratory for analysis.

Examination Protocol

Detailed specimen collection and processing instructions are described in the NHANES Laboratory/Medical Technologists Procedures Manual (LPM). Vials were stored under appropriate temperature conditions (stored at -20 degrees Centigrade) until they were shipped to Johns Hopkins University Lipoprotein Analytical Laboratory for testing. The analytical methods are described in the Analytic Methodology section of this document.

Data Collection

Detailed specimen collection and processing instructions are discussed in the LPM. Each chapter in the LPM specifies the procedure to be used for preparation, labeling, processing, preservation, and transport of the specimens.

Analytic Methodology

Total Cholesterol

Cholesterol is measured enzymatically in serum or plasma in a series of coupled reactions that hydrolyze cholesteryl esters and oxidize the 3-OH group of cholesterol. One of the reaction byproducts, H₂O₂ is measured quantitatively in a peroxidase-catalyzed reaction that produces a color. Absorbance is measured at 500 nm. The color intensity is proportional to cholesterol concentration. The reaction sequence is as follows:

                                        cholesteryl ester hydrolase
Cholesteryl ester + H₂O ------------------------------------->cholesterol + fatty acid

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

                                                                 peroxidase
2H₂O₂ + 4-aminophenazone + phenol ---------------------> 4-(p-benzoquinone
monoimino)-phenazone + 4 H₂O

HDL-Cholesterol

In NHANES 1999-2000, HDL-cholesterol was measured using two methods. A heparin-manganese (Mn) precipitation method and a direct immunoassay technique were used. The heparin-Mn method was used for participants ages 6 years and above. The direct method is used for participants ages 3-5 and for participants with no heparin-manganese HDL-cholesterol values, usually as a result of limited sample volume. HDL-cholesterol values less than 40 mg/dL are associated with increased coronary heart disease risk in adults.

Heparin-Mn Precipitation Method

Apolipoprotein B containing lipoproteins are removed by precipitation with heparin sulfate and MnCl₂ and cholesterol is measured in the HDL-containing supernatant. Cholesterol in the HDL-containing supernatant is measured as described above for total cholesterol.

HDL-Cholesterol Direct Immunoassay Method

HDL is measured directly in serum. The apolipoprotein B containing lipoproteins in the specimen are reacted with a blocking reagent that renders them non-reactive with the enzymatic cholesterol reagent under conditions of the assay. The reagents are purchased from Roche/Boehringer-Mannheim Diagnostics. The method uses sulfated alpha-cyclodextrin in the presence of Mg⁺² , which forms complexes with apoB containing lipoproteins, and polyethylene glycol-coupled cholesteryl esterase and cholesterol oxidase for the HDL-cholesterol measurement. The reactions are as follows:

ApoB containing lipoproteins + a-cyclodextrin + Mg⁺² + dextran SO₄ ---> soluble non-reactive complexes with apoB-containing lipoproteins 

                                     PEG-cholesteryl esterase
HDL-cholesteryl esters ------------------------------------> HDL-unesterified cholesterol + fatty acid

                                                PEG-cholesterol oxidase
Unesterified cholesterol + O₂ -------------------------------------> cholestenone + H₂O₂  

H₂O₂ + 5-aminophenazone + N-ethyl-N-(3-methylphenyl)-N’_succinyl ethylene diamine + H₂O + H⁺ peroxidase ---> qunoneimine dye + H₂O

Absorbance is measured at 600 nm. 

Analytical Note

Change in Assay Methods Most Likely Responsible for Changes in HDL Cholesterol values in NHANES 1999-2008

Researchers are cautioned to interpret trends in HDL cholesterol for NHANES 1999-2008 in view of probable HDL cholesterol method effects. The mean HDL cholesterol values from 1999-2008 showed changes for sample participants 20 years or older:

Mean HDL Cholesterol Values from 1999-2008

Years	n	Mean HDL(mg/dL)*	SEM**
1999-2000	4117	50.6	0.7
2001-2002	4691	51.9	0.28
2003-2004	4475	54.1	0.38
2005-2006	4481	54.6	0.36
2007-2008	5332	51.9	0.53

* Weighted mean to account for complex sample design of NHANES
**SEM is the standard error the mean

The changes in average HDL Cholesterol levels from 1999-2008 were seen across various age, gender and race-ethnicity groups indicating a method effect. The HDL cholesterol was analyzed in 1999-2002 using two methods - heparin manganese precipitation and a direct HDL immunoassay depending on the participant age and amount of specimen. Most participants in 1999-2002 were measured by the precipitation method. Starting in 2003, all HDL cholesterol samples were analyzed using the direct HDL cholesterol immunoassay method. The heparin-manganese precipitation method and direct immunoassay method for 1999-2000, 2001-2002 and 2005-2006 showed an undesirable bias (>4%) when compared to the laboratory's HDL-cholesterol quality controls (Solomon Park Research Laboratories, Kirkland, WA) that were assigned values established by the Centers for Disease Control and Prevention. The CDC HDL cholesterol reference method uses heparin-manganese to precipitate HDL-cholesterol and the Abell-Kendall method to measure cholesterol. The HDL cholesterol for 1999-2000, 2001-2002 and 2005-2006 were adjusted using:

Corrected HDL =       (Solomon Park assigned HDL value) x (Participant HDL)      
                            (Quality Control HDL value associated with Participant sample)

The bias for the HDL cholesterol method for 2003-2004 was acceptable (<4%) and the participant results were not corrected. In addition, there was a change in instrumentation in 2005-2006 and there were several modifications of the direct HDL cholesterol method. To control for these differences in methods and instrumentation, the HDL cholesterol was corrected using the Solomon Lab quality controls as described above. In 2007-2008, a new laboratory performed the HDL cholesterol using a direct HDL cholesterol method similar to the direct HDL method of 2005-2006, but it was performed on a different instrument.

Both laboratories performing HDL Cholesterol from 1999-2008 participated in the CDC-NHLBI Lipid Standardization Program (LSP). The CDC LSP maximum allowable bias for HDL Cholesterol is 5%. The average bias compared to the reference CDC HDL Cholesterol value for 2007-2008 was approximately -0.5%. The average bias compared to the CDC HDL Cholesterol values for 2003-2004 and 2005-2006 were approximately +2.7% and +2.0%, respectively. Unfortunately, the HDL Cholesterol values for the LSP program for 1999-2002 could not be evaluated. This indicates that the 2007-2008 HDL Cholesterol values may be less biased than the 2003-2006 values; however, both laboratories were within the 5% maximum allowable bias for HDL Cholesterol.

The changes in average HDL Cholesterol levels from 1999-2008 were seen across various age, gender and race-ethnicity groups indicating a method effect. Other covariates (body mass index, medications, physical exercise, smoking and alcohol consumption) may explain some of the changes in HDL cholesterol, but it is unlikely to account for the majority of the mean changes in HDL cholesterol.

Analytic Notes

LBXTC:

The Laboratory 13 data file contains laboratory test results for total cholesterol (LBXTC), which uses the reference analytic method. However, the NHANES Laboratory 18 biochemistry profiles also include measurements of total cholesterol (Laboratory 18 variable name: LBXSCH). In general, for most analyses, the appropriate variable to use is LBXTC.The value from the biochemistry profile (LBXSCH) should not be used routinely.

LBDTCSI:

The Total cholesterol in mg/dL (LBXTC) was converted to mmol/L (LBDTCSI) by multiplying by 0.02586.

LBDHDL:

HDL-cholesterol was derived from two HDL-cholesterol methods. See analytic methodology section above.

LBDHDLSI:

The HDL-cholesterol in mg/dL (LBDHDL) was converted to mmol/L (LBDHDLSI) by multiplying by 0.02586.

Sample Weights

Use the full sample weight (WTSMEC2YR) and the jackknife replicate sample weights (WTMREP01-WTMREP52) for serum total cholesterol and high density lipoprotein (HDL) cholesterol analyses.

The full sample weights are used to estimate means, percentages, medians and other percentiles, and regression coefficients.

The 52 jackknife replicate weights are used to estimate standard errors of these statistics.

Special Notes for this Dataset

The analysis of NHANES 1999-2000 laboratory data must be conducted with the key survey design and basic demographic variables. The NHANES 1999-2000 Household Questionnaire data files contain demographic data, health indicators, and other related information collected during the household components. The Household Questionnaire data files include all of the survey design variables and sample weights required to analyze these data. The Phlebotomy Examination file includes auxiliary information on duration of fasting, the time of day of the venipuncture, and the conditions precluding venipuncture. The Household Questionnaire and Phlebotomy Exam files may be linked to the laboratory data file using the unique survey participant identifier SEQN.

Codebook and Frequencies