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What are Biliverdin (BV) Isomers?
Biliverdin (BV) is a pivotal green pigment that emerges as a product of heme degradation, a vital biological process. Heme, an iron-containing molecule found in hemoglobin, undergoes catabolism primarily in the liver and spleen, where it is broken down into various metabolites. Biliverdin is one of the key intermediates in this pathway and serves as a precursor to bilirubin, the yellow pigment commonly associated with bruises and liver function tests.
Biliverdin itself exists in four primary isomeric forms, each distinguished by the specific methine bridge that has been cleaved from the heme molecule. These isomers are:
- Biliverdin IXα: This isomer is formed when the α-methine bridge in the heme ring is cleaved. It is the most commonly occurring form in many animals and plays a significant role in the metabolism of heme.
- Biliverdin IXβ: Resulting from the cleavage of the β-methine bridge, this isomer has distinct chemical properties compared to its α counterpart and is found in varying concentrations across different species.
- Biliverdin IXγ: This form is produced by removing the γ-methine bridge. It is less common than the α and β isomers but is important in understanding the full spectrum of biliverdin metabolism.
- Biliverdin IXδ: The δ-methine bridge cleavage produces this isomer, which also contributes to the overall biliverdin pool but is typically less prevalent.
Each biliverdin isomer has unique structural attributes that influence its chemical behavior and biological function. For instance, the different isomers can vary in their stability, solubility, and reactivity, which affects how they interact with other molecules and their roles in various physiological processes. The study of these isomers is crucial for understanding their specific contributions to heme metabolism, their potential health benefits, and their role in disease states.
Creative Proteomics utilizes advanced targeted metabolomics technology to provide precise analysis and identification of biliverdin isomers.
Biliverdin Isomers Analysis at Creative Proteomics
Separation and Identification: Using one-dimensional thin-layer chromatography (TLC) to effectively separate and identify biliverdin isomers (IXα, IXβ, IXγ, IXδ).
Biliverdin Quantification: Accurate measurement of the concentration of each biliverdin isomer in various biological and synthetic samples.
Custom Analysis: Tailored analytical services to meet specific research needs or diagnostic requirements.
Data Interpretation: Comprehensive analysis and interpretation of results, including detailed reports on biliverdin isomer profiles.
Analytical Techniques for Biliverdin Isomers Analysis
Thin-Layer Chromatography (TLC): Utilizes a TLC plate and solvent systems to separate biliverdin isomers based on their migration rates. This technique is used for initial separation and qualitative analysis.
High-Performance Liquid Chromatography (HPLC): Employs HPLC systems equipped with chromatographic columns and a solvent delivery system to achieve high-resolution separation of biliverdin isomers. Key instruments include:
- HPLC System: Agilent 1260 Infinity or similar.
- Chromatographic Columns: C18 reverse-phase columns for optimal separation.
Mass Spectrometry (MS): Coupled with HPLC for detailed analysis, MS instruments provide molecular identification and quantification of biliverdin isomers. Key instruments include:
- Mass Spectrometer: Thermo Scientific Q Exactive or similar.
- Ionization Source: Electrospray ionization (ESI) for optimal performance with HPLC.
Ultraviolet-Visible (UV-Vis) Spectroscopy: Measures absorption spectra of biliverdin isomers to support identification and quantification. Key instruments include:
- UV-Vis Spectrophotometer: PerkinElmer Lambda 365 or similar.
Sample Requirements for Biliverdin (BV) Isomers Analysis
| Sample Type | Recommended Volume |
|---|---|
| Serum | Minimum 1 mL |
| Bile | Minimum 1 mL |
| Plasma | Minimum 1 mL |
| Whole Blood | Minimum 1 mL |
| Tissues (homogenized) | Minimum 100 mg |
| Liver Biopsy | Minimum 100 mg |
| Urine | Minimum 5 mL |
| Feces | Minimum 100 mg (homogenized) |
| Eggshells | Minimum 100 mg (powdered) |
| Cell Cultures | Minimum 1 mL |
| Synthetic Solutions | Minimum 1 mL or 100 mg |
Report
- A detailed technical report will be provided at the end of the whole project, including the experiment procedure, instrument parameters.
- Analytes are reported as uM or ug/mg (tissue), and CV's are generally<10%.
- The name of the analytes, abbreviation, formula, molecular weight and CAS# would also be included in the report.
PCA chart
PLS-DA point cloud diagram
Plot of multiplicative change volcanoes
Metabolite variation box plot
Pearson correlation heat map
Simultaneous determination of free biliverdin and free bilirubin in serum: A comprehensive LC-MS approach
Journal: Analytica chimica acta
Published: 2024
Background
Bilirubin (BR) and biliverdin (BV) are key pigments in heme metabolism, with BR being a potent antioxidant and linked to reduced risks of cardiovascular diseases and diabetes. Measuring free BR and BV is challenging due to their complex properties and the limitations of current analytical methods, such as poor selectivity and sensitivity. Liquid chromatography-mass spectrometry (LC-MS) shows potential for accurate measurement but requires overcoming issues related to stability and isomerization.
Materials & Methods
Materials:
Chemicals: Biliverdin (BV) and bilirubin (BR) stock solutions (100 µM) were prepared in DMSO, sparged with argon, and further diluted. A DMSO₂O (1:1) mixture with 0.1 mg/mL ascorbic acid was used as a diluent.
Fetal Bovine Serum (FBS): Filtered through AMICON 10 kDa membranes; the third filtrate was used for analysis.
Sample Preparation:
Bilirubin Analysis: FBS filtrate (300 µL) was mixed with DMSO (300 µL) and ascorbic acid, then transferred into amber HPLC vials.
Biliverdin Analysis: FBS filtrate (300 µL) was acidified with formic acid, extracted with chloroform, evaporated, and reconstituted in a DMSO₂O mixture.
LC-MS/MS Method for Targeted Metabolomics
- Instrumentation: UHPLC Accela 1250 system coupled with LTQ Velos ion trap MS (Thermo Finnigan) with a heated ESI source in positive mode.
- Separation: Utilized a Kinetex C18 EVO column with a gradient of mobile phases comprising 5 mM ammonium formate (pH 3) and a mixture of 200 mM ammonium formate (pH 3), water, and ACN.
- MS Parameters: ESI heater temperature at 400°C, transfer capillary at 350°C, with specific voltages and gas settings. BV and BR were quantified using SRM transitions 583.2 → 297.2 and 585.2 → 299.0, respectively.
Validation:
The LC-MS/MS method was validated for precision, intermediate precision, linearity, detection and quantitation limits, selectivity, matrix effects, accuracy, and robustness.
Application:
The validated LC-MS/MS method was employed to analyze BV and BR in commercial serum samples, including gamma-irradiated and heat-inactivated FBS.
Results
Method Development: A C18-based UHPLC column with a 5 mM ammonium formate mobile phase allowed for baseline separation of BV, BR, and their isomers in 7.5 minutes. Positive ion mode LC-MS/MS was more sensitive than UV detection, with SRM mode providing the best results.
Separation of BV and BR (10 nM) along with their positional isomers found in commercially available standards. Chromatographic conditions are described in detail in Experimental section.
Analytical Challenges: BV and BR showed solubility issues in aqueous solvents, with DMSO or 50% DMSO (aq) being optimal. High aqueous content led to precipitation, while metal ion binding affected results. Stability varied: BR was more labile than BV, requiring protection from light and stabilization with ascorbic acid or argon sparging. EDTA caused bias by converting BR to BV.
Analyte loss as a function of solvent composition of standard solutions. Each standard solution was prepared by a 10x dilution of a corresponding solution in DMSO and was centrifuged prior to LC-MS/MS analysis. Data were normalized to DMSO at each concentration level.
Validation and Application: The method demonstrated high precision, accuracy, and sensitivity. It successfully differentiated BR and BV in human and bovine serum, revealing differences in their ratios and suggesting potential interspecies variations or effects of serum processing.
Table 1 LC-MS/MS method validation parameters
Reference
- Albreht, Alen, Mitja Martelanc, and Lovro Žiberna. "Simultaneous determination of free biliverdin and free bilirubin in serum: A comprehensive LC-MS approach." Analytica chimica acta 1287 (2024): 342073.
Why is it important to use specific chromatographic columns for BV isomer analysis?
The choice of chromatographic columns is crucial in BV isomer analysis because it directly impacts the resolution and accuracy of the separation process. Biliverdin isomers differ only slightly in their structure, so the stationary phase of the column must be highly selective to effectively distinguish between these closely related molecules.
C18 reverse-phase columns are commonly used because they provide excellent separation based on the hydrophobic interactions between the isomers and the stationary phase. The column's ability to achieve sharp, distinct peaks for each isomer is essential for accurate identification and quantification, particularly when analyzing complex biological samples where isomers may be present in low concentrations.
How does sample stability affect the analysis of Biliverdin isomers?
Sample stability is a significant factor in the analysis of Biliverdin isomers because these compounds are sensitive to environmental conditions such as light, temperature, and pH. Instability can lead to degradation or isomerization, where one isomer converts into another, complicating the analysis and potentially leading to inaccurate results.
To mitigate these issues, samples are typically stored and handled under controlled conditions. For instance, samples might be kept in dark, cool environments to prevent light-induced degradation. Additionally, using stabilizing agents or inert atmospheres (like argon) can help preserve the original isomeric composition until analysis. Proper sample handling ensures that the analysis reflects the true isomer distribution in the original biological material.
What role does mass spectrometry (MS) play in the identification of Biliverdin isomers?
Mass spectrometry (MS) is an essential tool in the identification and quantification of Biliverdin isomers. After separation by HPLC, the isomers are introduced into the mass spectrometer, where they are ionized, typically using electrospray ionization (ESI). The MS then measures the mass-to-charge ratio (m/z) of the ions, providing a molecular fingerprint that allows for the precise identification of each isomer.
MS is particularly valuable because it not only identifies the isomers based on their unique m/z values but also provides quantitative data, allowing for the determination of the concentration of each isomer in the sample. This dual capability makes MS a powerful technique for studying the complex profiles of Biliverdin isomers in biological systems.
How does the choice of solvent affect the separation of Biliverdin isomers?
The choice of solvent in HPLC plays a critical role in the separation of Biliverdin isomers. The solvent system, typically a mixture of water and an organic solvent like acetonitrile or methanol, influences the interactions between the isomers and the stationary phase of the chromatographic column.
A well-chosen solvent system will enhance the differences in these interactions, leading to better resolution of the isomers. For instance, a solvent with the right polarity can maximize the separation efficiency, ensuring that each Biliverdin isomer elutes at a distinct time, resulting in clear, well-separated peaks. Adjusting the solvent gradient is also crucial for fine-tuning the separation process, especially when dealing with closely related isomers like Biliverdin IXα and IXβ.
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