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What is Putrescine?
Putrescine, also known as tetramethylenediamine, is a foul-smelling organic compound with the chemical formula NH₂(CH₂)₄NH₂. Discovered by the Berlin physician Ludwig Brieger in 1885, putrescine belongs to a class of compounds known as biogenic amines, which also includes cadaverine. These compounds are produced during the breakdown of amino acids in both living and dead organisms. While putrescine and cadaverine are responsible for the characteristic odor of decomposing flesh, they are also implicated in a variety of other biological processes and conditions, including bad breath and bacterial vaginosis.
Putrescine is toxic at high doses, although in rats, its acute oral toxicity is relatively low at 2000 mg/kg body weight, with a no-observed-adverse-effect level of 180 mg/kg body weight. It is also found in semen and some microalgae, often accompanied by related molecules such as spermine and spermidine. Understanding the levels of putrescine in biological samples is crucial for various research and clinical applications.
Putrescine Analysis in Creative Proteomics
1. Quantitative Putrescine Analysis
We provide precise quantification of putrescine levels using advanced techniques like High-Performance Liquid Chromatography (HPLC) and Liquid Chromatography-Mass Spectrometry (LC-MS). This service ensures accurate measurement of putrescine concentrations in biological samples.
2. Putrescine Profiling
Our profiling service involves detailed analysis of putrescine and its derivatives. By employing Gas Chromatography-Mass Spectrometry (GC-MS) and other sophisticated methods, we offer comprehensive insights into putrescine and related biogenic amines.
3. Custom Method Development
We offer tailored analytical method development for unique research needs. This includes creating and validating methods for specific sample types or experimental conditions to ensure high accuracy and reliability.
4. High-Throughput Screening
Our high-throughput screening service allows for the simultaneous analysis of numerous samples, using automated systems to efficiently handle large datasets, ideal for extensive biochemical and drug discovery studies.
5. Consultation and Data Interpretation
We provide expert consultation and data interpretation services, offering detailed analysis reports and insights to help researchers understand their results and refine their research strategies.
6. Method Validation and Quality Control
We ensure that all analytical methods are rigorously validated and meet high standards of accuracy and reproducibility, adhering to stringent quality control protocols.
Technology Platforms Used for Putrescine Analysis
High-Performance Liquid Chromatography (HPLC)
HPLC is a fundamental technique for putrescine analysis due to its high sensitivity and resolution. This method involves the separation of putrescine from other components in a sample using a chromatographic column, followed by detection.
- HPLC System: Agilent 1260 Infinity II or Shimadzu LC-20AD
- Detector: UV-Vis Detector (e.g., Agilent 1260 Infinity II UV-Vis Detector)
- Precolumn Derivatization Reagent: Dinitrosalicylic Acid Chloride (DNS-Cl)
Advantages: Offers high sensitivity, reproducibility, and the ability to analyze complex mixtures.
Gas Chromatography-Mass Spectrometry (GC-MS)
GC-MS combines the separating power of gas chromatography with the detailed analysis capabilities of mass spectrometry. This technique is particularly useful for trace analysis of putrescine.
- GC System: Agilent 7890B or Thermo Scientific TRACE 1300
- MS System: Agilent 5977A or Thermo Scientific ISQ EC
- Derivatization: Use of derivatizing agents like N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA)
Advantages: Provides high sensitivity and detailed molecular information.
Liquid Chromatography-Mass Spectrometry (LC-MS)
LC-MS integrates liquid chromatography with mass spectrometry, allowing for the separation, identification, and quantification of putrescine in complex samples.
- LC System: Waters ACQUITY UPLC or Thermo Scientific Vanquish
- MS System: Waters Xevo TQ-S or Thermo Scientific TSQ Altis
Advantages: High sensitivity and specificity with minimal sample preparation.
Sample Requirements for Putrescine Analysis
| Sample Type | Volume | Storage Conditions | Additional Notes |
|---|---|---|---|
| Serum | 1-2 mL | Refrigerated (4°C) | Use anticoagulant-treated tubes to avoid clotting; minimize hemolysis |
| Tissue | 50-100 mg | Frozen (-80°C) | Avoid thawing and refreezing; handle samples with care to prevent degradation |
| Semen | 1 mL | Refrigerated (4°C) | Ensure proper collection and handling to preserve sample integrity |
| Microalgae | 1-5 mL | Frozen (-80°C) | Preserve sample immediately after collection to prevent degradation |
| Other | Variable | Consult with Creative Proteomics | Follow specific handling instructions based on sample type |
PCA chart
PLS-DA point cloud diagram
Plot of multiplicative change volcanoes
Metabolite variation box plot
Pearson correlation heat map
Polyamine metabolism impacts T cell dysfunction in the oral mucosa of people living with HIV.
Journal: Nature Communications
Published: 2023
Background
Polyamines, particularly putrescine, spermidine, and spermine, play critical roles in cellular processes such as growth, protein translation, and stress responses. HIV infection induces alterations in polyamine metabolism, leading to changes in T cell function and immune regulation. Previous studies have shown that polyamines contribute to T cell dysfunction and immune hyperactivation in HIV-infected individuals. This study explores the impact of polyamines on Th cell subsets, specifically focusing on TregDys (dysregulated Tregs) and Th17 cells, and examines the molecular mechanisms underlying these effects.
Materials & Methods
Human Samples
Gingival biopsies and saliva from healthy individuals and PLWH were collected with informed consent, approved by the University Hospitals Cleveland Medical Center Institutional Review Board. Tonsil samples, obtained from tonsillectomies, were processed for single-cell suspensions.
HTOC Cultures and HIV Infection
Tonsillar cells were cultured with TCR activating antibodies, TGF-β1, and IL-2, and then infected with HIV (X4-tropic or R5-tropic) using spinoculation. Cultures were expanded with or without specific inhibitors for 4–7 days.
Cell Culture Reagents and Inhibitors
TCR antibodies, cytokines, and inhibitors for polyamine synthesis and EIF5A hypusination were used. Lentiviral transduction was employed for ODC-1 knockdown.
Flow Cytometry
Cells were stained with fluorochrome-conjugated antibodies and analyzed for markers such as FOXP3 and IL-1β using flow cytometry. Controls included isotype and unstained samples.
Polyamines in cell lysates and supernatants were quantified using a fluorimetric method. Samples were processed to remove proteins before analysis, with concentrations normalized to viable cell numbers.
Salivary Metabolome Analysis
Saliva samples were analyzed by LC-MS after methanol extraction. UPLC and Q Exactive MS were used for metabolite quantification.
RNA Sequencing and Metabolome Data Analysis
RNA sequencing of gingival cells was performed to identify gene expression changes. Data were integrated with metabolome results, analyzed using statistical tests including t-tests and ANOVA.
Targeted Polyamine Quantification
Polyamines were quantified by LC-MS after derivatization with dansyl chloride, with analysis performed using UPLC-MRM/MS.
Statistical Analyses
Statistical significance was assessed using Prism 8 software with Mann–Whitney tests, ANOVA, and correlation analysis.
Results
Polyamine Effects on Th Cells:
Exogenous polyamines increase EIF5A expression and its hypusination, leading to TregDys cell induction and proliferation.
Polyamines upregulate EIF5A and hypusinated-EIF5A while downmodulating ODC-1 expression in CD4+ T cells.
Polyamines do not induce IFN-γ in FOXP3+ Tregs, but rather enhance FOXP3 expression in Th1-like cells (PD-1+IFN-γ+CD4+).
Polyamines increase AREG and KI-67 expression in TregDys cells, promoting their proliferation.
Impact on Th17 Cells and Mucosal Immune Activation:
HIV-infected patients show a reduced percentage of Th17 cells in the oral mucosa compared to healthy controls.
The TregDys/Th17 ratio is increased in the oral mucosa of people living with HIV (PLWH), correlating with elevated putrescine levels.
Polyamine levels, especially putrescine, are positively correlated with TregDys and Th hyperactivation in the oral mucosa.
Metabolic and Molecular Pathways:
HIV infection upregulates polyamine synthesis, affecting immune cell function and promoting TregDys and Th infidelity.
Alterations in amino acid metabolism, specifically nitrogen metabolism, and increased polyamine biosynthesis are observed.
Inhibition of ODC-1 or polyamine pathways impacts TregDys and Th17 cell populations but does not fully restore Th17 cells.
Clinical Implications:
The study identifies a potential negative-feedback loop where excessive polyamines repress ODC-1 expression during HIV infection.
The findings suggest that polyamine metabolism could be a target for therapeutic interventions aimed at modulating immune responses in HIV-infected individuals.
HIV-1 infection associated polyamine increase is dependent on ODC-1 activity.
HIV+ patients have skewed TregDys/Th17 ratios and CD4+ T cell hyperactivation correlating with salivary putrescine in the oral mucosa.
Reference
- Mahalingam, S. S., et al. "Polyamine metabolism impacts T cell dysfunction in the oral mucosa of people living with HIV." Nature Communications 14.1 (2023): 399.
What is the significance of measuring putrescine levels in biological samples?
Measuring putrescine levels is critical for various research and clinical applications. Putrescine is a biogenic amine involved in cellular processes, including cell proliferation and differentiation. Abnormal levels can indicate metabolic imbalances, presence of certain diseases, or biological changes. In clinical settings, elevated putrescine levels are associated with conditions like cancer, bacterial infections, and gastrointestinal disorders. Research applications include studying putrescine's role in metabolic pathways, aging, and neurobiology.
How do the different analytical techniques for putrescine analysis compare in terms of sensitivity and accuracy?
Each analytical technique offers unique strengths:
- HPLC: Provides high sensitivity and is excellent for separating putrescine from complex mixtures. It is highly reproducible and reliable but requires precolumn derivatization for optimal results.
- GC-MS: Offers very high sensitivity and specificity, particularly useful for trace analysis. It provides detailed molecular information but requires derivatization to make putrescine volatile.
- LC-MS: Combines the separation power of liquid chromatography with the detailed analysis capabilities of mass spectrometry. It offers high sensitivity and specificity with minimal sample preparation but can be more expensive and complex.
- Fluorometric Assays: High sensitivity and simplicity, ideal for quick and straightforward analysis, though less detailed than chromatographic or mass spectrometry methods.
- Colorimetric Assays: Useful for high-throughput screening with cost-effective and simple procedures, but typically less sensitive compared to HPLC or MS-based methods.
What are the common sources of error in putrescine analysis, and how can they be minimized?
Common sources of error include:
- Sample Contamination: Contaminants can interfere with accurate measurement. Use clean, appropriate containers and follow strict handling protocols to avoid contamination.
- Degradation of Samples: Putrescine can degrade if samples are not stored properly. Ensure proper refrigeration or freezing and minimize the time between sample collection and analysis.
- Instrumentation Calibration: Inaccurate calibration of instruments like HPLC or GC-MS can lead to errors. Regular calibration and maintenance of equipment are essential for reliable results.
- Derivatization Issues: For methods requiring derivatization, improper handling or reaction conditions can affect results. Ensure accurate reagent preparation and reaction conditions.
Minimizing these errors involves following standardized protocols, using well-maintained equipment, and adhering to best practices in sample handling and analysis.
Can putrescine levels be influenced by dietary intake or other environmental factors?
Yes, putrescine levels can be influenced by dietary intake and environmental factors. Diets rich in foods that contain or promote the production of biogenic amines, such as aged cheeses, fermented foods, or certain meats, can affect putrescine levels. Additionally, factors like stress, infection, or changes in gut microbiota can influence putrescine production and metabolism. When interpreting results, it is essential to consider these external factors and their potential impact on putrescine levels.
Polyamine metabolism impacts T cell dysfunction in the oral mucosa of people living with HIV.
Mahalingam, S. S., et al.
Journal: Nature Communications
Year: 2023
DOI: 10.1038/s41467-023-36163-2
Inflammation primes the kidney for recovery by activating AZIN1 A-to-I editing.
Heruye, Segewkal, et al.
Journal: bioRxiv
Year: 2023
DOI: 10.1101/2023.11.09.566426
