Table 1.
Characteristics of study population.
Figure 1.
Distribution of urine albumin and total protein levels in healthy donors.
A. Box-whisker and scatter plots of urine albumin distributions in urines. (Top panels: urine albumin concentration in mg/dL; Bottom panels: ratio of urine albumin to urine creatinine in mg/g). B. Box-whisker and scatter plots of urine total protein distributions in urine. (Top panels: urine total protein concentrations in mg/dL; Bottom panels: ratio of urine total protein to urine creatinine in mg/mg). Urine albumin, creatinine, and total protein were measured in urines obtained from 103 healthy donor by bead-based immunoassay, ELISA, and Bradford method, respectively. Boxes represent 25–75th percentiles, whiskers represent range.
Table 2.
Relative Abundance classes of urine protein biomarkers.
Figure 2.
Effect of several normalization methods on the population variability of urine proteins.
Urines obtained from 103 healthy donors were evaluated for levels of 211 proteins using multiplexed bead-based immunoassays. Coefficients of variation (CV) were determined for each of the 211 urine proteins based on absolute and normalized values. Correlation between the two sets of values was evaluated using Pearson's test of correlation. Normalized values were calculated by dividing absolute biomarker concentration by the level of several urine parameters: A. urine creatinine (UCr); B. urine albumin; C. urine total protein; D. ratio of urine albumin to urine creatinin (ACR); E. β-2-microglobulin.
Table 3.
Urine biomarkers correlating to specific urine parameters.
Figure 3.
Western blot of OPN, CA 125, HE4, and TTR from human urine.
A representative western blot of Osteopontin (OPN), CA125, HE4, and transthyretin (TTR) antigens expressed in protein lysates obtained from concentrated human urine samples from healthy individuals is depicted. Anti-Osteopontin, HE4, and TTR antibodies detected a single protein isoform of the corresponding proteins. Osteopontin migrated with an apparent molecular mass of 60 kDa, HE4 protein fragment migrated at 13 kDa, and TTR at 15 kDa. Anti-CA125 antibody detected three different protein fragments. The major CA125 fragment migrated in SDS-PAGE with an apparent molecular mass of 41 kDa (shown) and two smaller protein bands were in the range of ∼ 28–30 kDa. The presence of the different CA125 protein fragments in the human urine samples was confirmed by immunoblotting using various anti-CA125 antibodies. THP (68 kDa band) was evaluated as a loading control.
Figure 4.
Temporal variability of urine biomarkers.
Urines were collected three times a day (day, evening, night) over a two day period from 25 healthy female donors. Each urine sample was evaluated for 29 biomarkers by multiplexed immunoassay. Coefficients of variation (CV) were calculated for each biomarker among the three samples obtained each day (intra-day) and among the six samples obtained over the two day period (inter-day). Mean CVs for each reproducibly detectable biomarker in the entire 25 donor set are presented. Abs – absolute measurements; UCr – measurements normalized to levels of urine creatinine. Error bars represent 95% confidence intervals.