Common methods

Urine samples

Collecting 24 hour urine samples

Examples:

• UK NDNS surveys: Example method for study participants to take a 24 h urine sample Note: this method uses concomitant PABA administration. Adapt to purpose.
• US CDC: Details of a 24 h urine collection protocol for study participants using a questionnaire to assess urine collection completeness
• EUThyroid: instructions and guidance for taking urine samples is given on page 34 of this document prepared by the EUThyroid group. It relates to urine collection for iodine assessment, however can be adapted for use for other biomarkers. Urine sample handling advice is also included.

Addressing completeness of 24 h urine samples: Several methods are available to assess the completeness of 24 h urine collections:

• p-aminobenzoic acid (PABA) recovery (see below);
• Urinary creatinine concentration correction (see below);
• Questionnaire.

Questionnaire: Use of a questionnaire to assess 24 h urine collection completeness is reported in the US NHANES surveys (see link below). However, standardised procedures across study fieldworkers is paramount to reduce operator variation, and rigorous and intensive protocols are recommended to uphold data quality, including supervised urine collection. See this study for more details:

• Intersalt Cooperative Research Group 1988 Intersalt: an international study of electrolyte excretion and blood pressure. Results for 24 hour urinary sodium and potassium excretion. Intersalt Cooperative Research Group. BMJ 297:319-328.
• Method: Details of a 24 h urine collection protocol for study participants using a questionnaire to assess urine collection completeness, from US CDC.

24 hour urine samples vs spot urine samples

24 h urine samples are usually the gold standard for the measurement of most nutrient biomarkers. However, they can be cumbersome and require intensive and strict fieldwork protocols, and can introduce bias (i.e. from missed collection) in some settings.

Where collection of 24 h urine samples is impossible, spot urine samples may be a valuable alternative. Further, since spot samples are often routinely used in health surveys, e.g. for the measurement of urinary iodine, additional assay can be easily integrated into survey or research protocols. Spot samples also remove the need for multiple visits, and therefore may be a more efficient use of resources.

Example spot collection methods:
EUThyroid: instructions and guidance for taking urine samples is given on page 35 of this document prepared by the EUThyroid group. It relates to urine collection for iodine assessment, however can be adapted for use for other biomarkers. Urine samples handling advice is also included.Sample size estimates may need adjustment to provide the correct power and precision.See these selected references for example guidance on sample size:

• Andersen S, Karmisholt J, Pedersen KM, Laurberg P 2008 Reliability of studies of iodine intake and recommendations for number of samples in groups and in individuals. Br J Nutr 99:813-818.
• König F, Andersson M, Hotz K, Aeberli I, Zimmermann MB 2011 Ten repeat collections for urinary iodine from spot samples or 24-h samples are needed to reliably estimate individual iodine status in women. J Nutr 141:2049-2054.
• Eng J 2003 Sample size estimation: how many individuals should be studied? Radiology 227:309-313.

Urinary hydration adjustment methods

There are several ways to assess urinary hydration using proxy biomarkers, including creatinine (see below), specific gravity or osmolality. These are described in the following reference:

Middleton DR, Watts MJ, Lark RM, Milne CJ, Polya DA 2016 Assessing urinary flow rate, creatinine, osmolality and other hydration adjustment methods for urinary biomonitoring using NHANES arsenic, iodine, lead and cadmium data. Environ Health 15:68.

Creatinine concentration can be used to assess hydration, and is an alternative method for 24 h urine collection completeness assessment. Creatinine values should be interpreted dependent on sex, protein intake, muscle mass, degree of malnutrition and ethnicity. Since standard cut-offs do not widely exist, creatinine should be used with caution. 

Further reading on Creatinine:

• Schmidt RL, Straseski JA, Raphael KL, Adams AH, Lehman CM (2015) A Risk Assessment of the Jaffe vs Enzymatic Method for Creatinine Measurement in an Outpatient Population. PLOS ONE 10(11): e0143205.
• Delanghe JR, Speeckaert MM. Creatinine determination according to Jaffe-what does it stand for?. NDT Plus. 2011;4(2):83-6.
• Harry Husdan, Abraham Rapoport (1968) Estimation of Creatinine by the Jaffe Reaction. Clinical Chemistry 14 (3) 222-238;

Measurements of creatinine concentration and sodium concentration in a spot urine sample combined with details of the individual’s sex, weight, height and age allow application of the Kawasaki formula which estimates 24 h urine excretion (16). It is recommended that this method, which is less accurate than using 24 h collections, can be used for population assessments provided that the sample size of the group is adequate (5). Review the Kawasaki method.

For further reading:

Kawasaki T, Itoh K, Uezono K, Sasaki H 1993 A simple method for estimating 24 h urinary sodium and potassium excretion from second morning voiding urine specimen in adults. Clinical and experimental pharmacology & physiology 20:7-14.