• Component Description
• Eligible Sample
• Description of Laboratory Methodology
• Data Processing and Editing
• Laboratory Quality Assurance and Monitoring
• Analytic Notes
• Codebook
  • SEQN - Respondent sequence number
  • LB2DAY - Days between 1st and 2nd exam
  • LB2TC - Total cholesterol (mg/dL)
  • LB2TCSI - Total cholesterol (mmol/L)
  • LB2HDL - HDL-cholesterol (mg/dL)
  • LB2HDLSI - HDL-cholesterol (mmol/L)
  • LB2TR - Triglyceride (mg/dL)
  • LB2TRSI - Triglyceride (mmol/L)
  • LB2LDL - LDL-cholesterol (mg/dL)
  • LB2LDLSI - LDL-cholesterol (mmol/L)

National Health and Nutrition Examination Survey

2001-2002 Data Documentation, Codebook, and Frequencies

Cholesterol - Total, HDL, LDL & Triglycerides, Second Exam (l13_2_b)

Data File: l13_2_b.xpt

First Published: September 2010

Last Revised: NA

Component Description

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.

See the General Documentation on Second Day Laboratory Exams. Also, see the documentation for the primary exam data for Lab 13 and Lab 13AM.

Eligible Sample

Participants aged 16-69 who do not meet any of the exclusion criteria were sampled.

Description of Laboratory 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 2001-2002, 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 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.

Triglycerides

Triglycerides are measured enzymatically in serum or plasma using a series of coupled reactions in which triglycerides are hydrolyzed to produce glycerol. Glycerol is then oxidized using glycerol oxidase, and H2O2, one of the reaction products, is converted via peroxidase to a phenazone. Absorbance is measured at 500 nm. The reaction sequence is as follows:

                                         lipase
Triglycerides + 3H2O ----------------------> glycerol + fatty acids

                                    glycerokinase
Glycerol + ATP -----------------------------------> glycerol-3-phosphate + ADP

                                           glycerophosphate oxidase
Glycerol-3-phosphate + O2--------------------------------> dihydroxyacetone phosphate + H2O2

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

High levels of serum triglycerides help determine the risk for coronary heart disease (CHD) and peripheral atherosclerosis. High triglycerides are associated with increased risk for coronary artery disease (CAD) in patients with other risk factors, such as low HDL-cholesterol, some patient groups with elevated apolipoprotein-B, and patients with forms of low-density lipoprotein (LDL)-cholesterol that may be particularly atherogenic. Desirable fasting triglyceride levels are considered to be those below 150 mg/dL and are further categorized as borderline high, 150–199 mg/dL; high, 200–499 mg/dL; and very high, greater than or equal to 500 mg/dL. Very high triglycerides can result in pancreatitis. Triglycerides are also measured because the value is used to calculate LDL-cholesterol concentrations. Triglycerides are only reported for the primary sample for fasting (at least 8 hours or more but less than 24 hours) participants aged 16 and above who were examined in the morning sessions.

LDL-Cholesterol

Most of the circulating cholesterol is found in three major lipoprotein fractions: Very low-density lipoproteins (VLDL), LDL, and HDL. LDL-cholesterol is calculated from measured values of total cholesterol, triglycerides, and HDL-cholesterol according to the Friedewald calculation:

              [LDL-cholesterol] = [total cholesterol] – [HDL-cholesterol] – [triglycerides/5]

where [triglycerides/5] is an estimate of VLDL-cholesterol and all values are expressed in mg/dL. The calculation is valid only for triglycerides less than or equal to 400 mg/dL.

LDL carries most of the circulating cholesterol and, when elevated, contributes to the development of coronary atherosclerosis. LDL-cholesterol is measured to assess risk for CHD and to follow the progress of patients being treated to lower LDL-cholesterol concentrations. Desirable levels of LDL-cholesterol are below 130 mg/dL, borderline high is from 130–159 mg/dL, high is 160–189 mg/dL and very high LDL-cholesterol is greater than or equal to 190 mg/dL. LDL-cholesterol is only reported for the primary sample for fasting (at least 8 hours or more but less than 24 hours) participants aged 16 and above who were examined in the morning sessions. 

Data Processing and Editing

Blood specimens were processed, stored and shipped to Johns Hopkins University Lipoprotein Analytical Laboratory. Detailed specimen collection and processing instructions are discussed in the NHANES Laboratory/Medical Technologists Procedures Manual (LPM). Read the LABDOC file for detailed data processing and editing protocols. The analytical methods are described in the Description of Laboratory Methodology section.

Laboratory Quality Assurance and Monitoring

The NHANES quality control and quality assurance protocols (QA/QC) meet the 1988 Clinical Laboratory Improvement Act mandates. Detailed quality control and quality assurance instructions are discussed in the NHANES Laboratory/Medical Technologists Procedures Manual (LPM). Read the LABDOC file for detailed QA/QC protocols.

Analytic Notes

LB2TC:

The Laboratory 13 data file contains laboratory test results for Total Cholesterol (LB2TC), which uses the reference analytic method. However, the NHANES Laboratory 40 Biochemistry Profile also include measurements of Total Cholesterol (Laboratory 40 variable name: LB2SCH). In general, for most analyses, the appropriate variable to use is LB2TC.The value from the biochemistry profile (LB2SCH) should not be used routinely.

LB2TCSI:

The Total Cholesterol in mg/dL (LB2TC) was converted to mmol/L (LB2TCSI) by multiplying by 0.02586.

LB2HDL:

HDL-cholesterol was derived from two HDL-cholesterol methods. See Description of Laboratory Methodology above.

LB2HDLSI:

The HDL-cholesterol in mg/dL (LB2HDL) was converted to mmol/L (LB2HDLSI) by multiplying by 0.02586.

LB2TR: 

Triglyceride levels for the primary sample, were measured on examinees who were examined in the morning session only. The distribution of serum triglycerides for the primary sample should be estimated only on examinees ages 16 and above who fasted at least 8 hours or more but less than 24 hours, were examined in the morning, and were randomly assigned to the morning fasting sample.

The Laboratory 13AM Second Day Data File contains laboratory test results for Triglycerides (LB2TR) derived from the reference analytic method. However, the NHANES Lab 40 Biochemistry Profile also include measurements of triglycerides (Lab 40 variable name is LB2STR).  The appropriate variable to use is LB2TR from Lab 13AM.

LB2TRSI:

The Triglycerides value in mg/dL (LB2TR) was converted to mmol/L (LBDTRSI) by multiplying by 0.01129.

LB2LDL: 

LDL-cholesterol levels for the primary sample were measured on examinees that were examined in the morning session only. The distribution of serum LDL-cholesterol for the primary sample should be estimated only on examinees ages 16 and above who fasted at least 8 hours or more but less than 24 hours, were examined in the morning, and were randomly assigned to the morning fasting sample.  LDL-cholesterol is calculated from measured values of total cholesterol, triglycerides, and HDL-cholesterol according to the Friedewald calculation:

[LDL-cholesterol] = [total cholesterol] – [HDL-cholesterol] – [triglycerides/5]

where all values are expressed in mg/dL. The calculation is valid for triglycerides less than or equal to 400 mg/dL.

LB2LDLSI:

The LDL-cholesterol value in mg/dL (LB2LDL) was converted to mmol/L (LB2LDLSI) by multiplying by 0.02586.

Special Notes for this Dataset

The analysis of NHANES laboratory data must be conducted with the key survey design and basic demographic variables. The NHANES Household Questionnaire data files contain demographic data, health indicators, and other related information collected during the household interviews. 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 Examination files may be linked to the laboratory data file using the unique survey participant identifier SEQN.

Codebook and Frequencies