National Health and Nutrition Examination Survey

Second Day Exams

See the general documentation on Second Day laboratory exams. See the general documentation on second day laboratory exams. Also, see the documentation for the primary exam data for Laboratory 10 and 10AM.

Glucose, Insulin, C-peptide, and Glycohemoglobin

Diabetes mellitus was assessed by measures of plasma glucose, serum insulin, serum C-peptide, and blood glycohemoglobin in participants aged 16-69 years for a second day exam sample.

Diabetes is a leading cause of disease and death in the United States. Eight million Americans are known to have diabetes, and an equal number have undiagnosed diabetes. In 1993, nearly 18 percent of all deaths for persons over the age of 25 were among people with diabetes. The prevalence of diabetes and overweight (one of the major risk factors for diabetes) continue to increase. Substantial new efforts to prevent or control diabetes have begun, including the Diabetes Prevention Trial and the National Diabetes Education Program.

Information on the prevalence of diabetes disease, especially in its early stages, and associated risk factors will be used to help develop early intervention and prevention programs for the disabling consequences of this condition. Specifically, the diabetes disease examination will provide population data to: 1) determine a national estimate of diabetes disease prevalence (diagnosed and undiagnosed), including those at high risk for the late complications of the disease (i.e., ulceration and amputation); 2) identify the risk factors of diabetes disease; 3) permit a national cohort to be established for follow-up studies of this condition; and 4) provide critical information to clinicians and public health officials for the development of preventive care and community-based interventions.

Participants aged 16-69 years who do not meet any of the exclusion criteria are eligible.

Glucose

The enzyme hexokinase (HK) catalyzes the reaction between glucose and adenosine triphosphate (ATP) to form glucose-6-phosphate (G-6-P) and adenosine diphosphate (ADP). In the presence of nicotinamide adenine dinucleotide (NAD), G-6-P is oxidized by the enzyme glucose-6-phosphate dehydrogenase (G-6-PD) to 6-phosphogluconate and reduced nicotinamide adenine dinucleotide (NADH). The increase in NADH concentration is directly proportional to the glucose concentration and can be measured spectrophotometrically at 340 nm.

Insulin

Insulin radioimmunoassay (RIA) is a double-antibody batch method. Insulin in the specimen competes with a fixed amount of 125I-labelled insulin for the binding sites of the specific insulin antibodies. Bound and free insulin are separated by adding a second antibody, centrifuging, and decanting. The radioactivity in the pellet is then measured. The radioactivity is inversely proportional to the quantity of insulin in the specimen.

C-peptide

C-peptide radioimmunoassay (RIA) is a competitive assay where 125I-labelled C-peptide competes with C-peptide in the specimen for antibody sites. Bound and free C-peptide is separated by adding a second PEG-accelerated double antibody. The antibody-bound fraction is precipitated and counted. The radioactivity is inversely proportional to the quantity of insulin in the specimen.

Glycohemoglobin

Glycated proteins differ from non-glycated proteins by the attachment of a sugar moiety(s) at various binding sites by means of a ketoamine bond. Glycohemoglobin (GH) thus contains 1,2-cis-diol groups not found in non-glycated proteins. These diol groups provide the basis for separation of glycated and non-glycated components by boronate affinity chromatography 1,2,3. In this analytical technique, a boronate such as phenylboronic acid is bonded to the surface of the column support. When a solution of proteins (e.g. hemolysate) is passed through the column, the glycated component is retained by the complexing of its diol groups with the boronate. After the unretained non-glycated component elutes from the column, the glycated component is eluted from the column with a reagent that displaces it from the boronate.

The Primus instrument is a fully automated glycohemoglobin analyzer, which utilizes the principle of boronate affinity high performance liquid chromatography (HPLC) 4. The analytical column contains aminophenylboronic acid bonded to a porous polymer support (gel). The low- and high-pressure pumps transfer reagents through the analytical column, with reagent selection executed by a switching valve. Hemolyzed samples are automatically injected onto the column during the flow of A-Elution Reagent #1. The glycated component binds to the boronate, while the non-glycated component passes through the column to the spectrophotometric detector, where it is detected at wavelength of 413-±2 nm. After the elution of non-glycated component, the Primus instrument pumps B-Elution Reagent #2, which displaces the glycated component from the column. The glycated component then passes through the detector. In the final stage of each sample cycle, the column is re-equilibrated with Elution A-Reagent #1. All reagent selection occurs in a timed sequence designed to allow complete elution of non-glycated and glycated components.

Microprocessors (Model CLC330) or the PC computer (Model CLC385) control all functions in the liquid chromatograph and computing integrator. The signal from the spectrophotometric detector is processed and the concentration of glycohemoglobin is calculated as a percentage of the total detected. Integration is by peak area in millivolt-seconds. The chromatogram is plotted first as the signal is received by the detector. The raw % glycohemoglobin is calculated when glycated hemoglobin peak area is divided by the total hemoglobin peak area. Primus HPLC uses two point calibrators with HbA1c assigned values to obtain a final standardized glycohemoglobin. The Schiff base does not interfere with boronate affinity method.

Blood specimens were processed, stored and shipped to University of Missouri-Columbia, Columbia, Missouri for analysis. 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 the Laboratory Methodology section.

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.

NHANES 2000 (RDC) Laboratory Data

The second day exam data was a convenience sample and thus did not have sample weights. The analysis of NHANES 2000 laboratory data must be conducted with the key survey design and basic demographic variables. The NHANES 2000 Household Questionnaire Data Files contain demographic data, health indicators, and other related information collected during household interviews. They also contain all survey design variables required to analyze these data. The Phlebotomy Examination File includes auxiliary information such the conditions precluding venipuncture. The Household Questionnaire and Phlebotomy Examination File may be linked to the laboratory data file using the unique survey participant identifier SEQN.

LB2GLU and LB2GLUSI, Plasma glucose; LB2IN and LB2INSI, Insulin; LB2CP and LB2CPSI, C-peptide; LB2GH, Glycohemoglobin

Plasma glucose, serum insulin, serum C-peptide, and blood glycohemoglobin were measured by the Diabetes Diagnostic Laboratory at the University of Missouri-Columbia using Primus CLC330 (Primus Corporation, Kansas City, MO) on participants aged 16-69 years. The Boronate Affinity High Performance Liquid Chromatography (HPLC) system determines total glycohemoglobin by measuring 1,2-cis diol group found in glycated hemoglobin. The system has been standardized to the reference method used for the Diabetes Control and Complications Trial (DCCT). The affinity chromatographic method has demonstrated excellent, long-term precision (interassay CV's <3.0%) and is not affected by the presence of hemoglobin variants S, C, D and elevated HbF. The method is also less sensitive to hemoglobin degradation due to improper sample handling.
The Laboratory 10 and 10AM Second Day Data File contains laboratory test results for glucose (LB2GLU) measured using the reference analytic method. However, the Lab 18 Biochemistry profiles also included measurements of this analyte. The serum glucose values (LB2SGL) reported in this data release should not be used to determine undiagnosed diabetes or prediabetes. Instead, plasma glucose values (LB2GLU) should be used based on the reference analytic method of this analyte.

Conversion factor for insulin units:

The conversion factor for insulin is 1 uU/mL = 6.00 pmol/L. This unit conversion is based on the WHO standard adopted in 1987 based on a human insulin with a potency of 26,000 U/g⁵,⁶.

Data Access

All data are available through the NCHS Research Data Center.