Preparation of bilirubin standard solutions for assay calibration

doi:10.21203/rs.3.pex-1844/v1

Method Article

Preparation of bilirubin standard solutions for assay calibration

https://doi.org/10.21203/rs.3.pex-1844/v1

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Bilirubin (BR) is the product of cellular heme catabolism and the major bile pigment in animal blood. It is an established biomarker of hemolysis and liver function. In the last decade, mild hyperbilirubinemia has been shown to be a biomarker of lower cardiovascular disease risk, attributed to its antioxidant and anti-inflammatory effects. To use bilirubin as a predictive biomarker, new powerful methods are currently being developed for its direct analysis in human blood. To harmonize the different methods, it is essential to use high-quality BR standard solutions for assay calibration. We present here a protocol for the preparation of stable standard solutions in the range of 10^-9- 10^-5 M BR at pH 7.4 that can facilitate a universal approach to assay calibration.

Bilirubin

standard solutions

assay calibration

The pigment bilirubin is an endogenous compound formed in animals by heme catabolism, catalyzed by heme oxygenase and biliverdin reductase¹. Bilirubin is a linear tetrapyrrole in which two carboxyl groups form six intramolecular hydrogen bonds with nitrogen atoms that fix the molecule in its typical ridge-tile 3D configuration. Hydrogen bond-breaking solvents, especially dimethyl sulfoxide², dissolve bilirubin most effectively. In aqueous solution at a physiological pH of 7.4, the solubility of bilirubin decreases to 10^-9 M. In plasma, bilirubin ranges from 3 - 15 µM¹ and is almost completely bound to serum albumin (Kd = 17 nM)³.

Analysis of bilirubin in clinical laboratories is performed by automated methods⁴. For highly sensitive analysis of bilirubin in complex biological matrices, a number of other analytical methods have been introduced, based on HPLC and coupled with advanced detectors, various sensors, molecularly imprinted surfaces, or the fluorescent bilirubin-binding protein UnaG^5-7.

To harmonize the different methods used in experimental medicine and biology, it is important to use high-quality BR standard solutions for assay calibration, which could facilitate a universal approach to bilirubin analysis.

We present here a protocol for the preparation of stable standard solutions in the range of 10^-9 - 10^-5 M bilirubin determined by UV-vis spectroscopy (10^-5 M) or by the HUG-based fluorometric assay (10^-9 M)⁸.

TECHNICAL NOVELTIES

1. The standard solutions are in saline solution buffered at physiological pH (not in alkali, not in apolar solvents).

2. The standard solutions are in a wide range of concentrations.

3. The preparation has a quality control procedure based on direct analysis of bilirubin UV-VIS spectra or fluorescence emission of the HUG -bilirubin complex (no colorimetric reaction).

Ultrapure-water milliQ for the preparation of all solutions

Dimethyl sulfoxide (DMSO) (Sigma Aldrich, SHBM2271)

Sodium chloride (NaCl) (Sigma-Aldrich)

Potassium chloride (KCl) (Sigma-Aldrich)

Hydrochloric acid (HCl) (Sigma-Aldrich)

Sodium hydroxide (NaOH) (Sigma-Aldrich)

Potassium dihydrogen phosphate (KH₂PO₄) (Sigma-Aldrich)

Disodium hydrogen phosphate dihydrate (Na₂HPO₄·2H₂0) (Sigma-Aldrich)

Bilirubin (BR) (Sigma-Aldrich, B4126)

Bovine Serum Albumin, Fraction V (BSA) (SIGMA, A-7906)

HELP-UnaG (HUG) fusion protein, in-house production⁸

SOLVENTS

Acronyms and composition:

· DMSO: pure

· PBS: Phosphate Buffered Saline, (136.9 mM NaCl, 2.7 mM KCl, 10.0 mM, Na₂HPO₄ · 2H₂O, 1.8 mM KH₂PO₄, milliQ water), pH 7.4

· PBS-BSA: PBS containing 4 g·L^-1 BSA (60.6 µM BSA), pH 7.4

· PBS-BSA_dil: PBS containing 0.4 g·L^-1 BSA (6.06 µM BSA), pH 7.4

LABWARE

· Beaker (0.5 L)

· Duran Laboratory glass bottles (500 mL and 250 mL)

· Amber glass vials (10 mL)

· Eppendorf tubes (1.5 mL) (Sarstedt)

· Screw cap tube (15 or 50 ml) (Sarstedt)

· Tips (10 mL, 1 mL, and 200 µL) (Sartstedt)

· 96-well black polystirene plates (Nunc®)

· Gilson Pipetman Classic Variable Volume Pipette, 2-20 µL

· Gilson Pipetman Classic Variable Volume Pipette, 10-200 µL

· Gilson Pipetman Classic Variable Volume Pipette, 100-1000 µL

· Gilson Pipetman Classic Variable Volume Pipette, 1-10 mL

· Aluminium foil

· Lab Tube Racks

· Stainless steel spoon

· Laboratory tweezers stainless steel

· PTFE Magnetic Stirrer Bar

· Quartz cuvette l = 1cm

INSTRUMENTS

· Milli Q, Millipore Co., Bradford, MAMicrobalance (Mettler Toledo, XS205 Dual range)

· Magnetic stirrer (Icamag® Rec-G)

· pHmeter (Meterlab PHM240)

· Spectrophotometer (Cary 4E, Varian)

· Microplate reader (Synergy, BioTeck Winooski, VT)

· Mettler Toledo XS205 Balance.

PREPARATION OF BILIRUBIN STANDARD SOLUTIONS

Bilirubin (BR) solutions can be prepared in amber glass vials or in aluminum-coated screw cap tubes (Table 1).

Solution A

Weigh a few mg of the dry BR powder in an Eppendorf tube on a microbalance.

Add an appropriate volume of DMSO to obtain a 5 mM solution and vortex.

Store 20 µL aliquots of this solution in Eppendorf tubes at -20 °C.

Stability: 4 months at -20 °C.

Solution B1

Dilute 10 µL Solution A in 4990 µL PBS to obtain a 10 µM solution and vortex.

Dilute 500 µL Solution B1 in 4500 µL PBS to obtain a 1 µM solution and vortex.

Stability: <10 min. Use and analyze it immediately after preparation.

Solution B2

Dilute 10 µL Solution A in 4990 µL PSB-BSA 4 g·L^-1.

Dilute 500 µL Solution B2 in 4500 µL PBS to obtain a 1 µM solution and vortex.

Stability: 2 h at T = 25°C; wait at least 30 min before using this solution.

Standard Solutions C1

Dilute serial volumes of Solution B1 (1 and 10 µM) in PBS to a final volume of 5 mL.

The volumes are given in Table 1.

Stability: Use and analyse immediately after preparation (within 5 minutes).

Standard Solutions C2

Dilute serial volumes of Solution B2 (1 and 10 µM) in PBS-BSA 0.4 g·L^-1 to a final volume of 5 mL.

The volumes are given in Table 2.

Stability: Use and analyse the solution within 1 hour of preparation.

QUALITY CONTROL OF BILIRUBIN SOLUTIONS

1) UV-VIS Spectroscopy

o Add 3 mL Solution B1 or Solution B2 to a quartz cuvette (l = 1 cm).

o Prepare a set of n = 4 samples.

o Use PBS or PBS-BSA as blank.

o Record UV-VIS spectra in the range 350 < λ < 600 nm and find A_max = 440 nm and 465 nm for Solution B1 and B2, respectively.

o BR solutions should be considered accurate if the absorbance value is:

§ Solution B1: 0.446 (± 0.05) at λ_max = 440 nm

§ Solution B2: 0.636 (± 0.05) at λ_max = 465 nm

2) HUG-based Fluorometric assay ⁸

o Transfer 200 µL of Standard Solution C1 or C2 to a black 96-well plate containing 10 µL of 1 g/L (or 16.6 µM) HUG (final volume = 210 µL; final concentration HUG = 0.79 µM).

o Fill 5 wells for each BR concentration.

o Use 200 µL of the solvent of either C1 or C2 solutions (PBS or PBS-BSA_dil).

o Incubate the covered plate at room temperature (T = 25 °C).

o Reaction time:

1 hour for Standard Solution C1

2 hours for Standard Solution C2.

o Instrumental settings: λ_ex = 485 nm, λ_em = 528 nm, T = 25 °C, gain 100, reading height 2.50 mm.

3) Range of applicability

o 7< pH <10

o The same calibration curve is obtained at T = 25°C or 37°C

o BSA concentration does not affect the stability and solubility of BR in standard solution or the kinetics of binding with HUG in a wide range of concentrations (0.4 - 40 g·L^-1).

4) Data Analysis

o Calculate the mean ± standard deviation for each BR concentration (n = 5).

o Fit the data of standard curves by linear regression analysis.

The dissolution process of BR in physiological buffer is a critical requirement for biochemical assays. Many precautions must be taken when preparing aqueous BR solutions to avoid oxidative reactions and aggregation.

o Avoid direct light (work in dim light, use amber glass bottles; wrap all tubes and vessels in aluminum foil).

o Limit exposure to air, by plugging tubes immediately after adding solutions.

o Avoid excessive shaking, which leads to bilirubin aggregation.

o Thaw Solution A only once; do not reuse it.

o Perform all experiments with freshly prepared solutions, taking into account the time period during which the solutions are stable.

o BR solutions prepared in PBS (Solution B1) must be analyzed spectrophotometrically within 10 minutes

o BR solutions prepared in PBS-BSA (Solution B2) must be thoroughly mixed and analyzed after at least 30 minutes, the minimum time required to achieve signal stability.

o The actual concentration of 10 µM BR solutions can be checked by the diazo method ⁹.

o BR solutions (1 – 50 nM) in PBS (Solutions C1) are unstable and must be immediately mixed with HUG.

o The reaction temperature is defined as room temperature T = 25 ± 2°C.

o Cover 96-well plates during the reaction time, to prevent solvent evaporation and photo-degradation of bilirubin.

o Soak the glassware overnight in aqueous NaOH solution (0.1 M); rinse with aqueous HCl solution (0.1 M) followed by milliQ water.

o Do not re-use plastic tubes as BR may adsorb to the walls.

o Sources of error or deviation are:

§ Uncalibrated pipettes;

§ Inaccuracy in transferring concentrated solutions to other solvents when preparing dilutions, as some bilirubin may remain on the outer surface of the pipette tips;

§ Preparation of BR solutions at temperatures and times not specified above;

§ Exposure of standard solutions to light during preparation.

· From preparation of standard solution in PBS to starting incubation: 10 min

· From preparation of standard solution in PBS-BSA to starting incubation: 40 min

· Incubation time for standard solution in PBS: 1 h at 25°C

· Incubation time for standard solution in PBS-BSA: 2 h 25°C

· The overall time needed to complete the HUG-based fluorometric analysis: approximately 3 hours.

Identical standard solutions can be obtained by a single stock solution stored at -20° C for 4 months.

Standard solutions can be prepared in saline solutions buffered at physiological pH, spanning 10^-5 to 10^-9 M without or with BSA.

BR standard solutions without BSA have lower absorbance (Solution B1) and fluorescence (Solution C1) than identical solutions with BSA (Solutions B2 and C2).

BR standard solutions with BSA (Solutions B2 and C2) are stable at 25°C for 2 hours.

Assessment of BR standard solutions with BSA (Solutions B2) by UV-VIS spectroscopy correlates with the assessment by the diazo reaction ⁹.

1. Levitt, D. G. & Levitt, M. D. Quantitative assessment of the multiple processes responsible for bilirubin homeostasis in health and disease. Clin. Exp. Gastroenterol. 7, 307–28 (2014).

2. Brodersen, R. Bilirubin. Solubility and interaction with albumin and phospholipid. J. Biol. Chem. 254, 2364–2369 (1979).

3. Brodersen, R. Binding of bilirubin to albumin. CRC Crit. Rev. Clin. Lab. Sci. 11, 305–399 (1980).

4. Hulzebos, C. V et al. Diagnostic methods for neonatal hyperbilirubinemia: benefits, limitations, requirements, and novel developments. Pediatr. Res. (2021). doi:10.1038/s41390-021-01546-y

5. Ngashangva, L., Bachu, V. & Goswami, P. Development of new methods for determination of bilirubin. J. Pharm. Biomed. Anal. 162, 272–285 (2019).

6. Rawal, R. et al. A comprehensive review of bilirubin determination methods with special emphasis on biosensors. Process Biochem. 89, 165–174 (2020).

7. Narwal, V. et al. Bilirubin detection by different methods with special emphasis on biosensing: A review. Sens. Bio-Sensing Res. 33, 100436 (2021).

8. Bandiera, A. et al. Human elastin-like polypeptides as a versatile platform for exploitation of ultrasensitive bilirubin detection by UnaG. Biotechnol. Bioeng. 117, 354–361 (2020).

9. Klauke, R. et al. Reference measurement procedure for total bilirubin in serum re-evaluated and measurement uncertainty determined. Clin. Chim. Acta 481, 115–120 (2018).

Funded by Interreg V-A Italy-Croatia 2014-2020 (AdriAquaNet, ID 10045161, WP4.3 Welfare monitoring).

Ms. Paola Pelizzo (PhD student) and Ms. Luisa Donini (MSc student) contributed to the preparation of this protocol, as part of their training.

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Preparation of bilirubin standard solutions for assay calibration

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