- Service Details
- Demo
- Case Study
- FAQ
- Publications
What is Pyrimidine Biosynthesis?
Pyrimidine nucleotides play a critical role in cellular metabolism serving as activated precursors of RNA and DNA, CDP diacylglycerol phosphoglyceride for the assembly of cell membranes and UDP-sugars for protein glycosylation and glycogen synthesis. In addition, uridine nucleotides act via extracellular receptors to regulate a variety of physiological processes. There are two routes to the synthesis of pyrimidines; nucleotides can be recycled by the salvage pathways or synthesized de novo from small metabolites. Most cells have several specialized passive and active transporters that allow the reutilization of preformed pyrimidine nucleosides and bases.
The relative contribution of the de novo and salvage pathways depends on cell type and developmental stage. In general, the activity of the de novo pathway is low in resting or fully differentiated cells where the need for pyrimidines is largely satisfied by the salvage pathways. In contrast, de novo pyrimidine biosynthesis is indispensable in proliferating cells in order to meet the increased demand for nucleic acid precursors and other cellular components. Consequently, the activity of the de novo pathway is subject to elaborate growth state-dependent control mechanisms. Pyrimidine biosynthesis is invariably up-regulated in tumors and neoplastic cells, and the pathway has been linked to the etiology or treatment of several other disorders including AIDS, diabetes, and various autoimmune diseases such as rheumatoid arthritis.
Currently, a reliable and reproducible method using highly sensitive HPLC-MS platform for the rapid identification and quantification of pyrimidine biosynthesis metabolites in different sample types has been established by the experienced scientists at Creative Proteomics, which can satisfy the needs of academic and industrial study in your lab.
Figure 1. de novo pyrimidine biosynthesis.
Pyrimidine Biosynthesis Analysis in Creative Proteomics
Quantitative Analysis of Pyrimidine Metabolites
Creative Proteomics provides detailed quantitative analysis of pyrimidine metabolites using advanced techniques such as:
- Mass Spectrometry (MS): To measure the concentrations of pyrimidine nucleotides and their intermediates with high precision.
- High-Performance Liquid Chromatography (HPLC): For the separation and quantification of pyrimidine compounds from complex biological samples.
Metabolic Flux Analysis
This service focuses on tracking the flow of metabolites through the pyrimidine biosynthesis pathway. Techniques used include:
- Stable Isotope Tracing: Incorporating isotopic labels (e.g., carbon-13) into substrates to trace their incorporation into pyrimidine metabolites, providing insights into metabolic fluxes and pathway dynamics.
Pathway Mapping and Profiling
Creative Proteomics offers services for comprehensive mapping of the pyrimidine biosynthesis pathway, including:
- Pathway Reconstruction: Using analytical data to reconstruct the metabolic pathway and identify key intermediates and regulatory points.
- Metabolic Profiling: Analyzing the metabolic profile of cells or tissues to understand changes in pyrimidine metabolism under different conditions or treatments.
Custom Analysis and Consultation
Creative Proteomics also offers customized analysis and consultation services tailored to specific research or clinical needs. This includes designing bespoke experiments, interpreting complex data, and providing expert recommendations based on the analysis.
List of Pyrimidine Biosynthesis Metabolites We Can Analyze
| Pyrimidine Biosynthesis Metabolites Quantified in This Service | ||||
|---|---|---|---|---|
| Carbamoyl Phosphate | Aspartate | Carbamoyl Aspartate | Dihydroorotate | Orotate |
| Orotidine Monophosphate (OMP) | Uridine Monophosphate (UMP) | Uridine Diphosphate (UDP) | Uridine Triphosphate (UTP) | Cytidine Monophosphate (CMP) |
| Cytidine Diphosphate (CDP) | Cytidine Triphosphate (CTP) | Thymidine Monophosphate (TMP) | Thymidine Diphosphate (TDP) | Thymidine Triphosphate (TTP) |
| Deoxyuridine Monophosphate (dUMP) | Deoxythymidine Monophosphate (dTMP) | Deoxythymidine Diphosphate (dTDP) | Deoxythymidine Triphosphate (dTTP) | Orotic Acid |
Analytical Techniques for Pyrimidine Biosynthesis Analysis
High-Resolution Mass Spectrometer: Instruments such as the Orbitrap and Q-TOF provide high precision in identifying and quantifying pyrimidine metabolites.
Triple Quadrupole Mass Spectrometer: Used for targeted quantification and detailed analysis of specific pyrimidine compounds.
High-Performance Liquid Chromatography (HPLC)
HPLC System: Systems from brands like Agilent and Shimadzu are employed for the separation and analysis of pyrimidine metabolites.
Reverse-Phase Columns: For separating hydrophobic pyrimidine nucleotides and their derivatives.
Ion-Exchange Columns: For separating charged pyrimidine metabolites and their precursors.
Stable Isotope Mass Spectrometer: For tracing the incorporation of isotopically labeled substrates into pyrimidine metabolites.
Gas Chromatography-Mass Spectrometry (GC-MS): Complementary technique for analyzing metabolic fluxes and detecting low-abundance metabolites.
Sample Requirements for Pyrimidine Biosynthesis Analysis
| Sample Type | Recommended Sample Volume | Notes |
|---|---|---|
| Blood Plasma/Serum | 200-500 µL | Ensure samples are processed promptly to prevent degradation. |
| Cell Lysates | 100-300 µL | Cells should be harvested and lysed immediately. |
| Tissue Homogenates | 50-100 mg of tissue | Tissue should be rapidly frozen and stored at -80°C. |
| Urine | 1-5 mL | Samples should be collected in sterile containers and kept on ice. |
| Cell Culture Supernatants | 500 µL-1 mL | Ensure the supernatant is free of cell debris. |
| Biopsy Samples | 10-20 mg | Should be snap-frozen and stored at -80°C. |
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
Untargeted Metabolomics Based Prediction of Therapeutic Potential for Apigenin and Chrysin
Journal: International Journal of Molecular Sciences
Published: 2023
Background
Polyphenols, including apigenin and chrysin, are naturally occurring bioactive compounds found in various plant sources such as fruits, leaves, and vegetables. These compounds belong to the flavonoid family and have been associated with various health benefits, particularly in the context of cardiovascular disease, cancer, and neurodegenerative conditions. Despite their widespread use and recognized medical benefits, the specific pharmacological and toxicological effects of these compounds, especially when consumed through food in uncontrolled doses, are not fully understood. Previous research has shown that apigenin and chrysin can impact cellular signaling and function by regulating multiple targets. Recent studies have explored their roles in downregulating cholesterol biosynthesis while influencing other metabolic pathways, suggesting potential therapeutic applications, but also highlighting the need for careful evaluation of their risks and benefits.
Materials & Methods
1. Cell Culture and Treatments
- Cells: Mouse embryonic fibroblasts (MEFs) from Lonza (Cat# M-FB-481), passages 2-5.
- Medium: DMEM with high glucose (4.5 g/L), supplemented with 10% FBS and 1.5% penicillin/streptomycin.
- Treatment: Cells were cultured in serum-free DMEM with 1 g/L glucose. Treated with 25 μM apigenin or 25 μM chrysin for 24 hours. Samples were then frozen on dry ice for metabolomics analysis.
2. Sample Preparation
- Extraction: Cell pellets were thawed, covered with 80-85% methanol, and sonicated at 4°C for 30 minutes. Samples were then frozen at −40°C, vortexed, and centrifuged. The supernatant was prepared for LC-MS analysis.
UPLC-TOF-MS Analysis
- Equipment: Thermo UltiMate 3000LC with a hyper gold C18 column, coupled with Q Exactive mass spectrometry.
- Mobile Phase: Solvent A (0.1% formic acid, 5% acetonitrile) and Solvent B (0.1% formic acid, acetonitrile) with a gradient elution.
- Parameters: Flow rate of 0.3 mL/min, column temperature at 40°C. ESI+ mode with heater temperature at 300°C, spray voltage 3.0 kV, capillary temperature 350°C.
Statistical and Pathway Analysis
- Data Processing: Compound Discoverer 3.0 for alignment. Multivariate analysis with SIMCA-P (version 14.1) using PCA, PLS-DA, or OPLS-DA.
- Significance: VIP score > 1.5 and p-value < 0.05. Log2 fold change > ±1.5 considered significant. Data analyzed using GraphPad Prism.
Results
Apigenin and Chrysin Treatment Altered the Whole Cell Metabolome in MEF Cells
Apigenin and chrysin, two structurally related flavonoids, were found to significantly alter the metabolome of mouse embryonic fibroblast (MEF) cells. Using methods such as PCA, PLS-DA, and OPLS-DA, distinct clusters of metabolites were identified in cells treated with apigenin or chrysin compared to control cells. Volcanic plots highlighted the differential regulation of metabolites, with apigenin upregulating certain metabolite markers and chrysin downregulating others, confirming the significant impact of these flavonoids on the cellular metabolome.
IUPAC nomenclature for structurally related flavonoids. (A) Apigenin is a 5,7-Dihydroxy-2-(4′-hydroxyphenyl)-4H-chromen-4-one and (B) chrysin is a 5,7-Dihydroxy-2-phenyl-4H-chromen-4-one.
Alpha-Linolenic Acid and Linoleic Acid Metabolism Regulated by Apigenin
Apigenin specifically enriched pathways related to alpha-linolenic acid and linoleic acid metabolism. Significant increases were observed in metabolites such as eicosapentaenoic acid, docosapentaenoic acid, and adrenic acid in apigenin-treated MEF cells, suggesting apigenin's potential to enhance cardiovascular and cerebrovascular functions through these metabolic pathways.
Pathway enrichment analysis of altered metabolites in mouse embryonic fibroblasts following apigenin treatment. The negative ion mode results highlight the significant alteration of multiple metabolic pathways.
The pathway enrichment analysis and metabolic pathways significantly altered in positive ion mode following apigenin treatment.
Analysis of components of the alpha-linolenic acid pathway regulated by apigenin, showing significant increases in (A) eicosapentaenoic acid, (B) docosapentaenoic acid, and (C) docosahexaenoic acid in apigenin-treated MEFs.
Specific Pathways Regulated by Chrysin
Chrysin was found to regulate the alanine metabolism and urea cycle in MEF cells. Significant decreases in L-alanine, pyruvic acid, and lactic acid levels were observed, indicating chrysin's role in regulating energy metabolism and pyrimidine synthesis.
Regulation of alanine metabolism by chrysin, showing significant decreases in L-alanine, pyruvic acid, and lactic acid levels in MEFs.
Commonality in Downregulating Cholesterol and Uric Acid Biosynthesis
Both apigenin and chrysin demonstrated a similar ability to suppress cholesterol and uric acid biosynthesis pathways. They significantly reduced the levels of 7-dehydrocholesterol and xanthosine, suggesting their potential in lowering cholesterol and uric acid levels.
Downregulation of cholesterol and uric acid pathways by apigenin and chrysin, showing significant decreases in (A) 7-dehydrocholesterol and (B) xanthosine levels.
Reference
- Cochran, Cole, et al. "Untargeted metabolomics based prediction of therapeutic potential for apigenin and chrysin." International Journal of Molecular Sciences 24.4 (2023): 4066.
How do I prepare my samples for analysis?
Ensure samples are collected in appropriate containers, processed promptly, and stored under recommended conditions (e.g., snap-frozen tissue, kept on ice for urine).
What types of quality control measures are used?
We implement rigorous quality control procedures, including using high-resolution mass spectrometers and cross-validation with known standards to ensure accuracy and reliability.
What types of biological samples can you analyze?
We analyze a wide range of biological samples including blood plasma, serum, cell lysates, tissue homogenates, urine, cell culture supernatants, and biopsy samples.
Why is pyrimidine biosynthesis important for cells?
Pyrimidine biosynthesis is crucial for maintaining cellular functions because pyrimidine nucleotides are fundamental components of nucleic acids, which encode genetic information. Additionally, pyrimidine nucleotides play significant roles in other cellular processes, such as:
- Cell Membrane Formation: UDP (uridine diphosphate) is involved in the synthesis of glycolipids and glycoproteins, which are essential for cell membrane integrity and function.
- Protein Glycosylation: UDP-sugars are used in the glycosylation of proteins, which is vital for protein folding, stability, and function.
- Energy Transfer: ATP (adenosine triphosphate), though not a pyrimidine itself, is closely related and is used in energy transfer processes within the cell.
How does pyrimidine biosynthesis differ between proliferating and resting cells?
In proliferating cells, which are actively dividing and growing, the demand for nucleotides is high to support the synthesis of new DNA and RNA. Therefore, these cells primarily rely on the de novo synthesis pathway to produce pyrimidine nucleotides from scratch. In contrast, resting or differentiated cells, which are not actively dividing, have lower demands for new nucleotides and often utilize salvage pathways to recycle and reuse existing nucleotides, reducing the need for de novo synthesis.
NUDT22 promotes cancer growth through pyrimidine salvage and the TCA cycle.
Walter, M., Mayr, F., Hanna, B. M. F., Cookson, V., Mortusewicz, O., Helleday, T., & Herr, P.
Journal: Research Square
Year: 2022
https://doi.org/10.21203/rs.3.rs-1491465/v1
Untargeted metabolomics based prediction of therapeutic potential for apigenin and chrysin.
Cochran, C., Martin, K., Rafferty, D., Choi, J., Leontyev, A., Shetty, A., ... & Puthanveetil, P.
Journal: International Journal of Molecular Sciences
Year: 2023
https://doi.org/10.3390/ijms24044066
Cancer SLC43A2 alters T cell methionine metabolism and histone methylation.
Bian, Y., Li, W., Kremer, D. M., Sajjakulnukit, P., Li, S., Crespo, J., ... & Zou, W.
Journal: Nature
Year: 2020
https://doi.org/10.1038/s41586-020-2682-1
