Nanoscale Bilirubin Analysis in Translational Research and Precision Medicine by the Recombinant Protein HUG
Abstract
:1. Introduction
2. Results
2.1. Preparation and Quality Controls of Standard Bilirubin Solutions
2.1.1. Principles
2.1.2. Validation of Bilirubin Concentrations in Standard Solutions
2.1.3. Stability of Bilirubin Standard Solutions
- Stability of the 5 mM stock solution
- Stability of 10 µM pre-calibrator solutions
2.2. Nanoscale HUG-Based Fluorometric Assay of Bilirubin
2.2.1. Basic Requirements
- Concentration of HUG
- Concentration of BSA
2.2.2. Linear Range of the Fluorometric Assay
2.2.3. Optimal Reaction Time and Temperature
2.2.4. Limits of Nanoscale Bilirubin Detection and Quantitation
2.2.5. Solubility and Stability of Nanoscale Bilirubin Solutions
2.2.6. Accuracy and Precision
2.2.7. Robustness
2.3. Direct Analysis of Bilirubin in Human Plasma by the HUG Assay
3. Discussion
3.1. Advantages and Limitations of the HUG Assay
3.2. Domains of Application in Precision Medicine
4. Materials and Methods
4.1. Materials
4.2. Standard Bilirubin Solutions
4.2.1. Stock Solution
4.2.2. Pre-Calibrator Solutions
4.2.3. Calibrator Solutions
4.3. Spectrophotometric Measurements of Bilirubin Standard Solutions
4.4. Colorimetric Measurements of Bilirubin Standard Solutions
4.5. Fluorometric Measurements of Bilirubin Standard Solutions
4.6. Study Subjects and Ethics Statement
4.7. Bilirubin Fluorometric Measurements in Human Plasma Samples
4.8. DNA Extraction and Genotyping
4.9. Statistical Analyses
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Appendix A
Nominal Concentration (NC, nM) | Determined Concentration (DC, nM) | Relative Error, % | ||
---|---|---|---|---|
Day 1 | Day 2 | Day 1 | Day 2 | |
5 | 4.97 ± 0.10 | 5.19 ± 0.16 | 1 | −4 |
5 | 5.27 ± 0.11 | 5.16 ± 0.29 | −5 | −5 |
10 | 10.16 ± 0.34 | 10.83 ± 0.41 | −2 | −8 |
10 | 10.27 ± 0.11 | 10.85 ± 0.15 | −3 | −8 |
25 | 23.53 ± 0.48 | 27.32 ± 0.25 | 6 | −9 |
25 | 25.21 ± 0.20 | 25.45 ± 0.20 | −1 | −2 |
50 | 47.60 ± 0.22 | 49.14 ± 0.40 | 5 | 2 |
50 | 45.84 ± 0.38 | 50.27 ± 0.35 | 8 | −1 |
Nominal Concentration (NC, nM) | Determined Concentration (DC ± sd, nM) | Coefficient of Variation (%) | ||
---|---|---|---|---|
Day 1 | Day 2 | Day 1 | Day 2 | |
5 | 6.32 ± 0.37 | 4.83 ± 0.08 | 5.8 | 1.7 |
10 | 11.08 ± 0.46 | 10.07 ± 0.23 | 4.1 | 2.3 |
25 | 25.81 ± 0.49 | 24.92 ± 1.08 | 1.9 | 4.3 |
50 | 51.26 ± 1.21 | 50.10 ± 1.42 | 2.4 | 2.8 |
Sample | No Spike (0 nM) | Low Spike (5 nM) | Medium Spike (20 nM) | High Spike (50 nM) | |
---|---|---|---|---|---|
BSA 4 g × L−1 | 0 | 5.7 | 19.6 | 45.3 | |
1 | 11.8 | 6.3 | 18.3 | 38.2 | |
2 | 23.9 | 7.9 | 21.1 | 42.9 | |
3 | 10.4 | 4.5 | 19.1 | 36.7 | |
4 | 18.0 | 6.2 | 16.0 | 40.3 | |
5 | 17.1 | 5.3 | 17.5 | 41.4 | |
6 | 19.4 | 5.8 | 16.1 | 41.2 | |
7 | 28.0 | 4.9 | 17.3 | 45.1 | |
Sample (n) | Spike Level | Expected | Observed | Recovery % | p-value |
Plasma (7) | Low (5 nM) | 5.4 (±0.5) | 5.3 (±0.8) | 99.6 | p > 0.999 |
Med (20 nM) | 19.7 (±5.2) | 17.9 (±1.8) | 91.1 | p = 0.620 | |
High (50 nM) | 44.8 (±3.4) | 41.5 (±2.3) | 92.9 | p = 0.170 |
Key Features | Results | ||||
---|---|---|---|---|---|
1 | A priori, establish the acceptable limit of agreement | The limit of agreement should be <LoQ of the diazo method, which is the benchmark method. | |||
2 | The data structure | Paired measurements, each of them in four replicates; each measurement was in different subject. Data were obtained from a random sample of 36 human subjects. There was no prior knowledge of their total bilirubinemia, which was measured for the first time in this study by two methods. | |||
3 | Estimate the repeatability of the measures | HUG assay: RSD = 1%, n = 34; Diazo reaction: RSD = 5.3%, n = 34; Relative standard deviations of technical replicates. | |||
4 | Plot the data and inspect for absence of trend and constant variance | The dotted line represents Gaussian distribution. Shapiro–Wilk test for normal distribution accepted normality (p = 0.878). | |||
5 | If necessary, transform the data | Not needed. | |||
6 | Plot and report numerically the mean of the differences (bias) | −0.99 units (µM) | |||
7 | Give an estimate of the precision | The mean of the differences is −0.986; The standard deviation is 0.940. | |||
8 | Calculate and indicate the limits of agreement (LoAs) of the differences | Upper LoA = 0.86; Lower LoA = −2.83. | |||
9 | Provide an estimate of precision | 95% CI for mean of differences from −1.314 to −0.657. | |||
10 | Ensure that the range of the mean values is sufficient | The range of the mean total bilirubinemia values in our study group was 2.35–14.33 µM (HUG assay) or 2.12–16.78 µM (diazo assay), comprising the reference intervals of bilirubin concentrations in human sera [43], as well as the min–max range (1.5–22.6 µM) in the 226 subjects that were analyzed for group difference. | |||
11 | Variance between and within subjects | Inner confidence limits: there is a 2.5% probability that 95% of the differences lie between 0.518 and −2489 units (µM); Outer confidence limits: there is a 97.5% probability that 95% of the differences lie between 1.48 and −3.45 units (µM); Variance within subjects was RSD = 1% (HUG assay) and RSD = 5.3% (diazo reaction). | |||
12 | Software or computing processes used | Worksheet calculators attached to Carkeet’s ‘A Review of the Use of Confidence Intervals for Bland–Altman Limits of Agreement’, Optometry and Vision Science, January 2020. | |||
13 | Statistical assumptions made, such as normality of the data | No. | Statistic (W) | p-value | Decision at level (5%) |
34 | 0.976 | 0.651 | Cannot reject normality | ||
At the 0.05 level, the data were significantly drawn from a normally distributed population. |
SNP | Genotype | HWE | N (%) | Median (25–75%) | p-Value |
---|---|---|---|---|---|
BLVRA rs699512 | AA | 0.050 | 147 (65.6) | 5.53 (4.02–7.83) | Ref. |
GA | 63 (28.1) | 4.94 (3.54–6.84) | 0.191 | ||
GG | 14 (6.3) | 5.65 (5.18–6.56) | 0.815 | ||
GA + GG | 77 (34.4) | 5.42 (4.04–6.70) | 0.288 | ||
UGT1A1 rs8175347 * | –– | 0.823 | 85 (38.3) | 4.57 (3.77–5.98) | Ref. |
TA– | 106 (47.7) | 5.59 (4.02–7.36) | 0.008 | ||
TATA | 31 (14.0) | 9.38 (5.87–13.71) | <0.001 | ||
TA– + TATA | 137 (61.7) | 6.02 (4.31–8.07) | <0.001 | ||
HMOX1 rs2071746 | AA | 0.056 | 73 (32.6) | 5.23 (3.77–7.66) | Ref. |
TA | 97 (43.3) | 5.61 (4.28–7.77) | 0.195 | ||
TT | 54 (24.1) | 5.31 (4.03–6.98) | 0.755 | ||
TA + TT | 151 (67.4) | 5.57 (4.14–7.41) | 0.295 | ||
HMOX1 rs2071747 | GG | 0.122 | 210 (93.8) | 5.43 (3.97–7.47) 6.02 (4.42–7.23) | Ref. |
GC | 13 (5.8) | 6.02 (4.42–7.23) | 0.518 | ||
CC | 1 (0.4) | 2.37 (2.37–2.37) | 0.133 | ||
GC + CC | 14 (6,3) | 5.44 (4.34–7.13) | 0.816 | ||
HMOX2 rs2270363 ** | GG | 0.161 | 129 (57.8) | 5.37 (3.95–7.33) | Ref. |
GA | 76 (34.1) | 5.40 (4.02–7.53) | 0.956 | ||
AA | 18 (8.1) | 5.83 (4.35–7.83) | 0.520 | ||
GA + AA | 94 (42,2) | 5.57 (4.05–7.63) | 0.782 | ||
HMOX2 rs1051308 | AA | 0.346 | 113 (50.4) | 5.53 (3.95–7.75) | Ref. |
AG | 88 (39.3) | 5.26 (4.02–7.23) | 0.793 | ||
GG | 23 (10.3) | 5.53 (4.24–7.41) | 0.940 | ||
AG + GG | 111 (49,6) | 5.26 (4.02–7.28) | 0.845 |
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ID Term | [BR] | Solvent | Quality Control |
---|---|---|---|
A. Stock | 5 mM | DMSO | UV–VIS |
B. Pre-calibrator | 10 µM | PBS–BSA | UV–VIS; diazo reaction |
C. Calibrator | 50 nM | PBS–BSA | HUG fluorescence |
DMSO | PBS−BSA | PBS−HSA | PBS | |
---|---|---|---|---|
Amax (A.U.) | 0.631 (±0.02) | 0.636 (±0.05) | 0.601 (±0.02) | 0.446 (±0.05) |
λmax (nm) | 456 | 465 | 465 | 440 |
ε (cm−1 M−1) | 63,175 (±2859) | 61,079 (±1940) | 60,053 (±1425) | 41,885 (±2558) |
R2 | 0.9997 | 0.9992 | 0.9989 | 0.9946 |
LoD (nM) | 240 | 350 | 426 | 840 |
LoQ (nM) | 720 | 1100 | 1290 | 2600 |
[BSA] g × L−1 Pre-Calibrator | [BSA] g × L−1 Calibrators | Angular Coefficient (nM−1) |
---|---|---|
40 | 40 | 744 ± 74 |
4 | 4 | 815 ± 30 |
4 | 0.4 | 785 ± 36 |
BR (nM) | LoD (nM) | LoQ (nM) |
---|---|---|
0.05–10 | 0.36 | 1.10 |
0.5–50 | 1.56 | 4.75 |
Solvent | Angular Coefficient (nM−1) | R2 | Fluorescence Intensity of 50 nM BR Solution (A.U. ± SD) | Number of Replicates (n) |
---|---|---|---|---|
Tris 10 mM pH 8 | 663 | 0.9959 | 34,095 (±1189) | 5 |
HEPES 10 mM, pH 7.4 | 620 | 0.9911 | 31,313 (±2089) | 3 |
HBSS, pH 7.4 | 33 | 0.9841 | 1695 (±333) | 2 |
PBS pH 7.4 | 511 | 0.9994 | 25,390 (± 2868) | 40 |
PBS pH 8.5 | 523 | 0.9988 | 26,416 (± 1243) | 3 |
PBS pH 9.5 | 560 | 0.9985 | 28,172 (± 2484) | 3 |
PBS–Triton X–100 1%, pH 7.4 | 780 | 0.9965 | 40,045 (±2205) | 6 |
PBS–Triton X–100 1%, pH 8.5 | 750 | 0.9904 | 37,278 (±4863) | 6 |
PBS–Tween 20 1% | 7.5 | 0.9994 | 379 (±43) | 3 |
PBS–Na–Taurocholate 0.2 mM | 534 | 0.9999 | 26,662 (±1274) | 3 |
PBS–DMSO 30% | 757 | 0.9947 | 38,586 (±3190) | 3 |
PBS–0.4 g × L−1 BSA, pH 7.4 | 785 | 0.9990 | 39,950 (±1702) | 40 |
PBS–0.4 g × L−1 HSA, pH 7.4 | 718 | 0.9973 | 35,823 (±1862) | 3 |
Categorical Parameter | Median BR (µM) (25–75%) | p-Value | |
---|---|---|---|
Sex | Male | 5.88 (4.44–7.86) | <0.001 |
Female | 4.92 (3.31–6.36) | ||
UGT1A1 rs8175347 | –– | 4.57 (3.77–5.98) | Ref. |
TA– | 5.59 (4.02–7.36) | 0.008 | |
TATA | 9.38 (5.87–13.71) | <0.001 | |
TA– + TATA | 6.02 (4.31–8.07) | <0.001 |
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Sist, P.; Tramer, F.; Bandiera, A.; Urbani, R.; Redenšek Trampuž, S.; Dolžan, V.; Passamonti, S. Nanoscale Bilirubin Analysis in Translational Research and Precision Medicine by the Recombinant Protein HUG. Int. J. Mol. Sci. 2023, 24, 16289. https://doi.org/10.3390/ijms242216289
Sist P, Tramer F, Bandiera A, Urbani R, Redenšek Trampuž S, Dolžan V, Passamonti S. Nanoscale Bilirubin Analysis in Translational Research and Precision Medicine by the Recombinant Protein HUG. International Journal of Molecular Sciences. 2023; 24(22):16289. https://doi.org/10.3390/ijms242216289
Chicago/Turabian StyleSist, Paola, Federica Tramer, Antonella Bandiera, Ranieri Urbani, Sara Redenšek Trampuž, Vita Dolžan, and Sabina Passamonti. 2023. "Nanoscale Bilirubin Analysis in Translational Research and Precision Medicine by the Recombinant Protein HUG" International Journal of Molecular Sciences 24, no. 22: 16289. https://doi.org/10.3390/ijms242216289