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Cadaverine Analysis Service

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What is Cadaverine?

Cadaverine, also known by its chemical names 1,5-pentanediamine or pentamethylenediamine, is a foul-smelling diamine compound that arises primarily from the breakdown of proteins in animal tissues during putrefaction. This toxic substance was first reported by Ludwig Brieger, a Berlin-based physician, in 1885. Cadaverine is formed through the decarboxylation of the amino acid lysine, resulting in a molecule with the chemical formula NH₂(CH₂)₅NH₂. Structurally similar to putrescine, cadaverine's noxious odor and toxic nature make it a compound of interest in various fields, including toxicology, biochemistry, and medical research.

Biological Formation of Cadaverine

Cadaverine is produced when lysine undergoes decarboxylation, which involves the removal of a carboxyl group (COOH) from lysine, leaving an amine group (NH₂) attached to the fifth carbon in the lysine molecule. The result is a diamine that is both poisonous and irritating to the skin. Cadaverine poses health risks if ingested, inhaled, or absorbed through the skin, as it is highly destructive to mucous membranes and can cause burns. Although it exhibits low oral toxicity in rodent models, intravenous administration of cadaverine in rats leads to a dose-dependent decrease in blood pressure, and high dietary levels have been linked to reduced body weight and diminished food intake.

The significance of cadaverine extends beyond its role in tissue decomposition. Elevated cadaverine levels are found in individuals with lysine metabolism deficiencies, and the compound also serves as a precursor in the production of high polymers, adding importance to its study in biological research.

Creative Proteomics offers targeted metabolomics analysis services to facilitate qualitative and quantitative analysis of cadaverine.

Cadaverine Analysis in Creative Proteomics

Quantification of cadaverine in biological samples:

We specialize in detecting and quantifying cadaverine in biological matrices such as serum, tissues, urine, and cell cultures. Our methods ensure accurate and consistent results across different sample types.

In vitro and in vivo cadaverine monitoring:

Our services support both in vitro and in vivo studies, providing detailed analysis of cadaverine levels in laboratory and preclinical research settings.

Biogenic amines profiling:

In addition to cadaverine, we can analyze other biogenic amines involved in various biological and pathological processes, offering a comprehensive view of amine profiles.

Forensic and toxicological analysis:

We assist in forensic investigations by analyzing cadaverine in decomposing tissues, aiding research on tissue decay and postmortem intervals.

Food spoilage detection:

We help monitor cadaverine levels in food products like meat and fish to assess spoilage, ensuring food quality and safety.

Metabolic disorder research support:

Elevated cadaverine levels are associated with certain metabolic disorders, such as lysine metabolism deficiencies. We provide accurate analysis to support research and diagnostics in this area.

Analytical Techniques for Cadaverine Analysis

High-Performance Liquid Chromatography (HPLC)

HPLC is one of the most widely used methods for cadaverine analysis due to its precision and ability to separate complex mixtures. In our workflow, we often employ precolumn derivatization with reagents like DNS-Cl (1-fluoro-2,4-dinitrobenzene) to enhance the detection of cadaverine and other biogenic amines. This approach offers:

  • High sensitivity for detecting cadaverine in small concentrations.
  • Accurate quantification with high reproducibility.
  • Compatibility with various sample types, including serum, tissues, and food products.

Gas Chromatography-Mass Spectrometry (GC-MS)

For volatile or semi-volatile compounds like cadaverine, gas chromatography coupled with mass spectrometry (GC-MS) is a powerful alternative to HPLC. This technique provides:

  • High specificity: The mass spectrometer allows for precise identification of cadaverine based on its unique molecular structure.
  • High sensitivity: GC-MS is capable of detecting extremely low levels of cadaverine, making it suitable for trace analysis.
  • Separation of complex mixtures: GC efficiently separates cadaverine from other volatile compounds, ensuring accurate measurements.

Liquid Chromatography-Mass Spectrometry

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) offers a highly sensitive and selective approach for cadaverine analysis, particularly in more complex biological samples. This method allows:

  • Simultaneous detection and quantification of multiple biogenic amines, including cadaverine.
  • Enhanced specificity through tandem mass spectrometry, where two rounds of mass selection improve the precision of detection.
  • Wide dynamic range, making it suitable for both low and high concentration measurements in various matrices.

Sample Requirements for Cadaverine Analysis

Sample TypeVolume/WeightStorage ConditionsPreparation Notes
Serum100 µL-80°CShip on dry ice
Tissue≥50 mg-80°CFreeze immediately after collection
Urine500 µL-80°CPre-filter large particles
Cell Culture≥1 x 10⁶ cellsFreeze in liquid nitrogenWash in PBS before freezing
Food Samples≥10 gRefrigerate at 4°C, or freeze at -20°CGrind and homogenize if necessary
Principal Component Analysis (PCA) chart showing the distribution of samples across principal components

PCA chart

Partial Least Squares Discriminant Analysis (PLS-DA) point cloud diagram illustrating the separation of sample groups in a multidimensional space

PLS-DA point cloud diagram

Volcano plot depicting multiplicative changes in metabolite levels, highlighting statistically significant variations

Plot of multiplicative change volcanoes

Box plot showing the variation in metabolite levels across different sample groups, indicating median, quartiles, and outliers

Metabolite variation box plot

Pearson correlation heat map representing the correlation coefficients between different variables, with a color gradient indicating the strength of correlations

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

The study investigates how viruses alter the metabolic states of immune cells, particularly focusing on the mechanisms behind persistent immune dysfunction during chronic viral infections like HIV. Despite advances in combined anti-retroviral therapy (cART), HIV infection is associated with systemic inflammation, persistent mucosal immune dysfunction, and an increased risk of biological aging and related comorbidities. People living with HIV (PLWH) face a heightened risk of periodontitis, oral candidiasis, and various cancers. Previous studies indicated that HIV infection leads to dysregulation and hyperactivation of T helper (Th) cells in the oral mucosa, with specific focus on dysfunctional FOXP3+ Tregs and Th17 cells. This study explores how polyamine metabolism, particularly through ornithine decarboxylase (ODC-1) and eukaryotic translation initiation factor 5A (EIF5A) hypusination, influences Th cell balance and immune dysfunction in the oral mucosa of PLWH.

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.

Polyamine Assay

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

Impact on T Cell Subsets:

HIV-1 infection significantly increases the frequency of TregDys cells (FOXP3+ PD-1+ IFN-γ+) in the CD4+ T cell population.

There is a marked reduction in Th17 cells in the oral mucosa of HIV+ patients, even after cART therapy.

Effects of Polyamines:

Polyamines elevate EIF5A and its hypusinated form while decreasing ODC-1 expression in CD4+ T cells.

Exogenous polyamines do not induce TregDys cells by increasing IFN-γ levels; instead, they promote FOXP3 expression in Th1-like cells and enhance TregDys proliferation.

Spermidine and other polyamines contribute to the dysregulation of Th1-like cells and increase TregDys expansion, with a corresponding rise in Amphiregulin (AREG) and KI-67 expression.

Correlation with Oral Mucosa:

The ratio of TregDys to Th17 cells is significantly higher in the oral mucosa of people living with HIV compared to healthy controls.

Elevated levels of putrescine in saliva positively correlate with increased TregDys/Th17 ratios and Th cell hyperactivation in the oral mucosa.

Overall Summary:

HIV-1 infection induces a shift in T cell subsets, increasing TregDys cells and reducing Th17 cells, which is linked to elevated polyamine levels.

Polyamines upregulate EIF5A and promote TregDys proliferation while inhibiting IFN-γ expression in CD4+ T cells.

Elevated putrescine levels and increased TregDys/Th17 ratios in the oral mucosa of HIV+ individuals further support the role of polyamines in T cell dysregulation and immune hyperactivation.

Salivary metabolome analysis in conjunction with transcriptome and flow cytometry analysis of gingival immune cellsSalivary metabolome analysis in conjunction with transcriptome and flow cytometry analysis of gingival immune cells
Figure A: Circos plot linking dysregulated genes in PLWH to KEGG pathways. Figure B: Bar graph showing enrichment of terms by -log10(p-value) from dysregulated genes and metabolites. Figure C: Arginine-proline metabolism pathway with metabolite and gene changes (Log2FC). Figure D: Flow cytometry histograms of intracellular ODC-1 in CD4+ T cells from controls and PLWH. Figure E: Statistical analysis of ODC-1 fluorescence intensity in CD4+ T cells, showing median ± SEM. Figure F: Flow cytometry histograms of intracellular EIF5A in CD4+ T cells from controls and PLWH. Figure G: Statistical analysis of EIF5A fluorescence intensity in CD4+ T cells, showing median ± SEM.

Reference

  1. 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 factors can affect the accuracy of cadaverine measurements in biological samples?

The accuracy of cadaverine measurements can be influenced by several factors, including sample handling, storage conditions, and matrix complexity. Proper sample preservation is critical, as cadaverine can degrade or react with other compounds if not stored at the correct temperature, typically at -80°C for biological samples like serum or tissue. In addition, matrix effects such as interference from other biogenic amines or proteins in the sample can affect the precision of detection. At Creative Proteomics, we use techniques like precolumn derivatization to minimize interference and improve sensitivity, ensuring more accurate results.

How does the presence of other biogenic amines, like putrescine, affect cadaverine analysis?

Biogenic amines, such as putrescine, share similar chemical structures with cadaverine and can co-occur in biological samples, especially during tissue decomposition or in cases of metabolic imbalance. This can lead to cross-interference in the analysis, especially when using methods like HPLC or GC-MS. However, Creative Proteomics employs advanced separation techniques, including mass spectrometry (LC-MS/MS), to differentiate between closely related amines and accurately quantify cadaverine, even in complex samples with high levels of other amines.

Can cadaverine levels be used as a biomarker for specific diseases or conditions?

Yes, elevated cadaverine levels are often associated with certain metabolic disorders, particularly those related to lysine metabolism deficiencies. In clinical research, monitoring cadaverine concentrations can help diagnose or track diseases involving abnormal amino acid metabolism. Additionally, cadaverine can be used as a marker in forensic and toxicological studies to assess postmortem decomposition stages. Creative Proteomics provides customized cadaverine analysis to support disease research and forensic applications, ensuring accurate biomarker detection.

How do environmental factors influence cadaverine formation, and can they affect analysis results?

Environmental factors such as temperature, humidity, and oxygen levels can significantly impact the rate of protein decomposition and cadaverine formation in tissues. For instance, higher temperatures accelerate the putrefaction process, leading to more rapid cadaverine production, while anaerobic conditions can alter the metabolic pathways of bacterial breakdown. These environmental influences need to be considered when interpreting cadaverine levels in forensic or food spoilage studies. At Creative Proteomics, we provide context-based interpretation of cadaverine data, considering environmental conditions that may affect formation rates.

What are the challenges in detecting cadaverine in food products, and how are they addressed?

Detecting cadaverine in food products, particularly in meat and fish, can be challenging due to the complexity of the food matrix and the presence of other spoilage-related amines. The fat content, proteins, and water content in food can interfere with detection methods. At Creative Proteomics, we overcome these challenges by using advanced sample preparation techniques such as homogenization and pre-filtration, along with highly sensitive detection methods like GC-MS. This ensures accurate detection of cadaverine, even in complex food matrices, helping assess food safety and spoilage levels.

What are the detection limits for cadaverine in different sample types, and how does Creative Proteomics ensure sensitivity?

Detection limits for cadaverine can vary depending on the sample type and the analytical technique used. For example, LC-MS/MS and GC-MS offer extremely low detection limits, often in the nanomolar range, making them ideal for detecting trace levels of cadaverine in biological fluids or tissues. To ensure high sensitivity, Creative Proteomics uses precolumn derivatization for HPLC and mass spectrometric techniques that enhance the detection of even the smallest quantities of cadaverine. This ensures that we can accurately measure cadaverine levels, even in samples with low concentrations.

How are cadaverine levels in in vitro studies relevant to human in vivo conditions?

Cadaverine levels in in vitro studies, such as those using cultured cells or tissues, can offer valuable insights into metabolic pathways, enzyme activity, and cellular response to lysine decarboxylation. However, translating these findings to in vivo human conditions requires careful consideration of factors such as systemic metabolism, organ-specific responses, and interactions with other biomolecules. At Creative Proteomics, we help researchers correlate in vitro data with potential in vivo implications, offering a comprehensive understanding of cadaverine's role in biological systems.

Polyamine metabolism impacts T cell dysfunction in the oral mucosa of people living with HIV.

Mahalingam, S. S., Abigail Klug, Wiebke Thormann, Anita Parmar, Sean T. Scibelli, Fabiola Gonzalez, Vanessa Reinhardt, et al.

Journal: Nature Communications

Year: 2023

https://doi.org/10.1038/s41467-023-36163-2

* For Research Use Only. Not for use in diagnostic procedures.
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