Variable Urine Dilution

Although 24-hour urine samples or multiple specimens collected over time are preferred for exposure assessment of polar chemicals or metabolites, spot or convenience urine samples are most commonly collected. Spot samples, even if they are first morning voids, vary in dilution depending on the hydration state of the participant at the time of sample collection, which can, in turn, affect the concentration of the toxicant measured. Inter- and intra-individual variability in urinary concentrations of chemicals can be affected by urine dilution level, among several other factors.

Traditional approaches for correcting for urine dilution factor are creatinine correction, specific gravity correction, calculation of urinary excretion rate, using creatinine or specific gravity as an independent variable in a model that evaluates toxicant exposure and outcome, or use of directed acyclic graphs.1-3 Osmolality is an alternate method but is not routinely used, and there is presently no proficiency testing program or recommended method to support osmolality measurements.

  • Creatinine, a byproduct of muscle (or protein) catabolism, is typically excreted at a relatively constant rate (~±10%) within a healthy individual but varies widely when major physiologic changes such as body building, weight loss/gain, or pregnancy are taking place. Creatinine excretion differs with respect to factors such as race/ethnicity, age, sex, lean muscle mass or body mass index (BMI), and physiologic changes in pregnancy.1 Creatinine correction of urine dilution should be used cautiously and with consideration of creatinine-dependent factors of the population being studied (e.g., race, age, sex). Creatinine correction may artificially increase or decrease chemical/metabolite concentrations if creatinine-dependent factors are not appropriately considered. For example, consider a 6-year-old child and a 30-year-old adult who have the same concentration of an analyte in their urine and the same hydration state. Comparing their creatinine-corrected analyte values would be inappropriate because a child would excrete about half the amount of creatinine daily as an adult, making it appear that the child’s value is twice that of the adult.
  • Specific gravity, the measure of dissolved solids in urine, is often correlated to the creatinine concentration, but because it has less resolution, it is not as highly affected by demographic factors and so is often used instead of creatinine correction.4,5 However, it is normalized on the median specific gravity of the population rather than a constant value, and this may hinder the ability to compare concentrations across populations.2,3
  • Use of creatinine or specific gravity as a covariate in statistical models is common but may pose problems, especially if the target toxicant(s) of concern physiologically interacts with creatinine, as with arsenic or melamine.1

It is also worth noting that underlying disease conditions, especially kidney diseases, can affect creatinine excretion rate. Also, controversies surround the approach of correction for urine dilution in reporting chemical concentrations.3 We recommend exploring which, if any, approach is best for each population/analyte combination with perhaps multiple approaches explored and reported to enable comparisons across data sets. Summary statistics should be reported for values both corrected and uncorrected for dilution. When correcting or adjusting for creatinine or specific gravity, potential biases should be addressed, for example using sensitivity analyses that exclude creatinine from the model and that remove extreme dilute or concentrated urine values.

NOTE: Additional sample volumes are required for measurement of either creatinine and/or specific gravity.

  • Calculate creatinine-corrected toxicant concentration by dividing the toxicant concentration in ng/mL (µg/L) units by creatinine in mg/dL units and multiplying by 100 to give units µg/g creatinine units.
  • Calculate specific gravity–corrected toxicant concentration using the formula (final units are ng/mL):

    Pc = P[(SGm − 1)/(SG − 1)], where

    Pc is the specific gravity-corrected toxicant concentration (ng/mL),

    P is the observed toxicant concentration (ng/mL),

    SGm is the median SG value among the study population, and

    SG is the specific gravity of the individual urine sample.

  • Calculate the urinary excretion rate (UER) by multiplying the toxicant concentration in urine (Cu) by the volume of the entire urine void delivered from the bladder (Vu) and dividing by the time that the void was accumulating in the bladder (collection time (tc) – time of last urination (tl)). The UER would be used as the urine dilution value in data analysis.

1. Barr DB, Wilder LC, Caudill SP, et al. Urinary creatinine concentrations in the U.S. population: implications for urinary biologic monitoring measurements. Environmental Health Perspectives. 2005;113(2):192-200.

2. O'Brien KM, Upson K, Buckley JP. Lipid and creatinine adjustment to evaluate health effects of environmental exposures. Current Environmental Health Reports. 2017;4(1):44-50.

3. O'Brien KM, Upson K, Cook NR, et al. Environmental chemicals in urine and blood: improving methods for creatinine and lipid adjustment. Environmental Health Perspectives. 2016;124(2):220-227.

4. Meeker JD, Barr DB, Ryan L, et al. Temporal variability of urinary levels of nonpersistent insecticides in adult men. Journal of Exposure Analysis and Environmental Epidemiology. 2005;15(3):271-281.

5. Meeker JD, Hu H, Cantonwine DE, et al. Urinary phthalate metabolites in relation to preterm birth in Mexico City. Environmental Health Perspectives. 2009;117(10):1587-1592.

6. Muscat JE, Liu A, Richie J Jr. A comparison of creatinine vs. specific gravity to correct for urinary dilution of cotinine. Biomarkers. 2011;16:206-211.

7. Xia Y, Wong LY, Bunker BC, et al. Comparison of creatinine and specific gravity for hydration corrections on measurement of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine. Journal of Clinical Laboratory Analysis. 2014;28(5):353-363.

8. Gamble MV, Hall MN. Relationship of creatinine and nutrition with arsenic metabolism. Environmental Health Perspectives. 2012;120(4):A145-A146.

9. Gamble MV, Liu X. Urinary creatinine and arsenic metabolism. Environmental Health Perspectives. 2005;113(7):A442; author reply A442-A443.

10. Peters BA, Hall MN, Liu X, et al. Creatinine, arsenic metabolism, and renal function in an arsenic-exposed population in Bangladesh. PloS One. 2014;9:e113760.

11. Zhu H, Kannan, K. Inter-day and inter-individual variability in urinary concentrations of melamine and cyanuric acid. Environment International. 2019;123:375-381.