National Health and Nutrition Examination Survey

The 24-Hour Urine Collection component was added to NHANES in 2014 to: 1) estimate dietary intakes of sodium and potassium; 2) estimate the excretion of microalbumin (albumin and creatinine); and 3) assess measures of kidney markers (phosphorus, magnesium, calcium, oxalate, and urea nitrogen), caffeine, volatile organic compounds, and iodine in the U.S. population.

In 2014, a random half sample of non-pregnant participants aged 20-69 years and examined in the mobile examination center (MEC) were asked to collect their urines for a 24-hour period. For the first half of the year, a random half of those who completed the initial 24-hour urine collection were recruited to collect a second 24-hour urine specimen 3 to 10 days later. For the second half of the year, all those who completed the first 24-hour urine collection were recruited to collect a second 24-hour urine specimen. Data processing information from the initial and second 24-hour urine collections are released in two separate datasets (UR1_H_R and UR2_H_R). Each of these two files contain information on: total urine volume, length of collection, completeness of the urine collection, number of complete urine specimens, collection day of the week, and responses to questions on the participants’ experience collecting the urine to assess completeness of the specimen. Please see the documentation for these two files for more details.

Separate datasets were produced to include laboratory results of analytes from the 24-hour urine collections. The present file contains analyte data for urine kidney markers (albumin, creatinine, calcium, magnesium, oxalate, phosphorus, and urea nitrogen). See Appendix I for the list of data files for the 24-hour urine collection.

Urinary kidney markers are a diverse set of analytes often used to measure many complex and serious medical conditions including: obesity, renal disease, cardiac disease, hypertension, diabetes and hepatic disease. Each analyte provides unique information that may provide greater insight into many of the challenging public health issues that are prevalent in the U.S. Additionally, some of these kidney markers also aid in the assessment of nutritional status. When used in conjunction with measurements of dietary sodium over a 24-hour period, kidney marker measurements may aid in providing enhanced understanding of how to efficiently address many of these growing public health concerns.

To reduce the risk of inadvertent disclosure, all data from this 1-year 24-hour urine collection can only be accessed through the NCHS Research Data Center (RDC). Instructions for requesting use of these data are available at the RDC website (https://www.cdc.gov/rdc/).

The eligible sample was a random one-half sample of all examined participants aged 20-69 years with a few participants eliminated based on exclusion criteria (e.g. pregnant). Among participants with a complete 24-hour urine specimen, a portion of them were eligible for collection of a second 24-hour urine specimen. For the first half of the year, a random one-half were asked to collect a second specimen. For the second half of the year, all of the eligible participants were asked to collect a second 24-hour urine specimen.

1. Albumin

A fluorescent immunoassay for the measurement of human urinary albumin is described by Chavers et al. The methodology involves solid-phase, non-competitive, double-antibody reaction. Urine specimen albumin antigen reacts with albumin antibody that is covalently attached to polyacrylamide beads. This resulting solid-phase antibody complex is then reacted with fluorescein-labeled antibody. Unattached fluorescent antibody and other proteins are removed by centrifugation. The fluorescence of the stable solid-phase double-antibody complex is measured with a fluorometer and is directly proportional to the amount of urine albumin present. The standard line calibration material is human serum albumin with a range of 0.5 to 20 µg/mL.

2. Creatinine

In this enzymatic method, creatinine is converted to creatine under the activity of creatininase. Creatine is then acted upon by creatinase to form sarcosine and urea. Sarcosine oxidase converts sarcosine to glycine and hydrogen peroxide, and the hydrogen peroxide reacts with chromophore in the presence of peroxidase to produce a color product that is measured at 546 nm (secondary wavelength = 700 nm). This is an endpoint reaction that agrees well with recognized HPLC methods, and it has the advantage over Jaffe picric acid-based methods that are susceptible to interferences from non-creatinine chromogens.

3. Phosphorus

This method utilizes ammonium molybdate as the color-forming reagent. Measurement of the final product occurs at 340 nm (secondary wavelength 700 nm). Inorganic phosphate forms an ammonium phosphomolybdate complex having the formula (NH4)3[PO4 (MoO3)12] with ammonium molybdate in the presence of sulfuric acid. The concentration of phosphomolybdate formed is directly proportional to the inorganic phosphate concentration.

4. Magnesium

In this method magnesium reacts with Chlorophosphonazo III (CPZ III) and causes an increase in absorbance. EGTA is added to inhibit calcium interference. Addition of EDTA to the reaction allows for accurate sample blanking. The reaction is a two-point, end-point method that is measured photometrically at 660 nm.

5. Calcium

In this method calcium reacts with 5-nitro-5’-methyl-BAPTA (NM-BAPTA) in an alkaline environment to form a complex that is measured at 340nm. When EDTA is added to the reaction, two new complexes form, and the change in absorbance is directly proportional to the calcium concentration.

6. Oxalate

This method is an enzymatic process, based on the oxidation of oxalate by oxalate oxidase, followed by measurement of hydrogen peroxide produced during the reaction by a peroxidase-catalyzed reaction. The primary measuring wavelength is 600 nm, and the secondary measuring wavelength is 700 nm. The procedure is specific for oxalate.

7. Urea Nitrogen

This method utilizes a coupled enzyme reaction (urease, followed by glutamate dehydrogenase), with measurement of NADH (converting to NAD+) occurring at 340 nm.

Refer to the Laboratory Method Files section for detailed laboratory procedure manual(s) of the methods used.

Albumin - Kidney Markers - 24 Hour Urine - 1st Collection (October 2015)

Creatinine - Kidney Markers - 24-Hour Urine - 1st Collection (October 2015)

Urine specimens were processed, stored, and shipped to University of Minnesota, Minneapolis, MN for analysis. Detailed instructions on specimen collection and processing are discussed in the NHANES 24-Hour Urine Study Procedures Manual. Vials are stored under appropriate frozen (-30°C) conditions until they are shipped to University of Minnesota for testing.

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

Mobile Examination Centers (MECs)

Laboratory team performance is monitored using several techniques. NCHS and contract consultants use a structured quality assurance evaluation during unscheduled visits to evaluate both the quality of the laboratory work and the quality-control procedures. Each laboratory staff member is observed for equipment operation, specimen collection and preparation; testing procedures and constructive feedback are given to each staff member. Formal retraining sessions are conducted annually to ensure that required skill levels were maintained.

Analytical Laboratories

NHANES uses several methods to monitor the quality of the analyses performed by the contract laboratories. In the MEC, these methods include performing blind split samples collected on “dry run” sessions. In addition, contract laboratories randomly perform repeat testing on 2% of all specimens.

NCHS developed and distributed a quality control protocol for all the contract laboratories which outlined the use of Westgard rules (Westgard et al., 1981) when running NHANES specimens. Progress reports containing any problems encountered during shipping or receipt of specimens, summary statistics for each control pool, QC graphs, instrument calibration, reagents, and any special considerations are submitted to NCHS quarterly. The reports are reviewed for trends or shifts in the data. The laboratories are required to explain any identified areas of concern.

The data were reviewed. Incomplete data or improbable values were sent to the performing laboratory for confirmation.

Variables with data from 24-hour urine first collection and second collection specimens are denoted with a “UR1” and “UR2” prefix, respectively.

Refer to the 2013 - 2014 Laboratory Data Overview for general information on NHANES laboratory data.

Subsample weights

NHANES participants were selected on the basis of a national probability design.  In order to increase the number of participants for specific demographic groups, a multi-stage, unequal probability of selection design was implemented. In 2014, the sample design included an oversample of certain demographic groups: persons 80 years and older, non-Hispanic black, non-Hispanic Asian, Hispanic, and low income persons. Sample weights are constructed that encompass the unequal probabilities of selection, as well as adjustments for non-participation by selected sample persons. In order to produce national, representative estimates, the appropriate sample weights must be used.

The 24-hour urine collection was conducted for a random one-half subsample of 1,103 persons 20-69 years examined in the mobile examination center in 2014. A special one-year urine sample weight (WT1YUR) is provided for the 827 participants who provided a complete 24-hour urine specimen to obtain representative national estimates based on this first 24-hour urine collection. As with other NHANES subsamples, the 24-hour urine sample weights account for the additional probability of selection into the subsample component as well as the additional nonresponse. This sample weight, along with strata (VSTRA) and PSUs (VPSU) are used to calculate variance estimates based on the Taylor Series Linearization method. In addition, 16 Fay-adjusted balanced repeated replication (Fay’s BRR) weights (WT1YUB01 – WT1YUB16), along with their corresponding strata (VSTRABRR) and PSUs (VPSUBRR), and 15 Jackknife replicate weights (WT1YUR01 – WT1YUR15) that can be used with VSTRA and VPSU are included in the files to obtain variance estimates based on a replication method. The Fay’s BRR weights were created with an adjustment factor of 0.3. There is no separate sample weight provided for use with the data collected for the second 24-hour urine specimen. These data were collected for the purpose of estimating within-person variability and not for obtaining nationally representative estimates based on the second 24-hour urine sample. New sample weights would need to be calculated by the user if any additional analytical efforts were planned for the second collection data.

Demographic and Other Related Variables

The analysis of NHANES laboratory data must be conducted using the appropriate survey design and demographic variables. The NHANES 2013-2014 Demographics File contains demographic data, health indicators, and other related information collected during household interviews.

Please see Appendix 1 for the list of data files produced from the 24-hour urine collection.

This laboratory data file can be linked to the other NHANES data files using the unique survey participant identifier (i.e., SEQN).

Detection Limits

The detection limits were constant for all of the analytes in the data set. Two variables are provided for each of these analytes. The variable name ending in “LC” (ex., UR1MALC) indicates whether the result was below the limit of detection: the value “0” means that the result was at or above the limit of detection, “1” indicates that the result was below the limit of detection.  For analytes with analytic results below the lower limit of detection (ex., UR1MALC=1), an imputed fill value was placed in the analyte results field. This value is the lower limit of detection divided by the square root of 2 (LLOD/sqrt[2]). The other variable prefixed UR1 (ex., UR1MA) provides the analytic result for that analyte.

The lower limit of detection (LLOD) for 24-Hour Urine Kidney Markers First and Second Collections:

Please refer to the NHANES Analytic Guidelines and the on-line NHANES Tutorial for further details on the use of sample weights and other analytic issues.