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What is Spermidine?
Spermidine is a naturally occurring polyamine that plays a critical role in various biological processes, particularly within cellular metabolism and growth. Structurally, spermidine is composed of three amino acids—arginine, methionine, and cysteine—linked together to form a unique compound essential for cell function. It is found in all living organisms, with significant concentrations in various tissues, including the brain, liver, and kidneys.
Spermidine is involved in numerous key cellular functions that underpin health and longevity. One of its primary roles is to promote cell proliferation by stabilizing DNA and RNA structures, which facilitates vital processes such as protein synthesis and cell division. This function is especially important for tissue regeneration and repair. Additionally, spermidine enhances autophagy, the cellular mechanism that removes damaged organelles and proteins, thereby maintaining cellular health and homeostasis. This process is crucial in aging cells, where the accumulation of waste can lead to dysfunction. Furthermore, spermidine acts as a signaling molecule that helps cells respond to oxidative stress, promoting resilience and protecting against various age-related diseases, including neurodegenerative disorders and cardiovascular conditions.
Emerging studies suggest that spermidine may offer several health benefits, making it a topic of great interest in both scientific research and clinical applications. It has garnered attention for its potential anti-aging properties, with studies in model organisms demonstrating that spermidine supplementation can extend lifespan by promoting autophagy and cellular repair mechanisms. Research indicates that spermidine may have protective effects on neuronal cells, potentially reducing the risk of neurodegenerative diseases such as Alzheimer's and Parkinson's. Additionally, spermidine has been linked to improved metabolic health, including enhanced insulin sensitivity and reduced inflammation, which can significantly prevent metabolic disorders such as obesity and type 2 diabetes.
At Creative Proteomics, we offer specialized spermidine analysis services to support cutting-edge research into its biological functions and potential applications in health and disease management.
Spermidine Analysis in Creative Proteomics
Spermidine Quantification: We provide precise measurement of spermidine levels in biological matrices, including blood, urine, and tissue samples. Our methodologies utilize advanced techniques such as High-Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS) for high sensitivity and specificity.
Metabolite Profiling: We offer comprehensive profiling of spermidine and its metabolites, enabling researchers to explore metabolic pathways and interactions with other biomolecules for deeper insights into its biological functions.
Custom Analysis Services: We tailor spermidine analysis services to meet unique project requirements, whether for targeted sample analysis or developing specific protocols.
Spermidine Stability Testing: Our stability testing evaluates how spermidine levels fluctuate under different storage conditions and over time, ensuring sample integrity for experimental design.
Data Interpretation and Consultation: We provide expert consultation to help interpret results, discuss findings, suggest follow-up experiments, and leverage data for further research.
Quality Control and Assurance: We prioritize quality, with all services undergoing rigorous quality control measures to ensure reproducible and reliable results, adhering to strict industry standards.
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Metabolomics Services
We provide unbiased non-targeted metabolomics and precise targeted metabolomics services to unravel the secrets of biological processes.
Our untargeted approach identifies and screens for differential metabolites, which are confirmed by standard methods. Follow-up targeted metabolomics studies validate important findings and support biomarker development.
Download our brochure to learn more about our solutions.
Technology Platforms Used for Spermidine Analysis
High-Performance Liquid Chromatography (HPLC): This technique allows for the precise separation and quantification of spermidine from complex biological matrices.
Mass Spectrometry (MS): Coupled with HPLC, mass spectrometry offers high sensitivity and specificity in detecting and quantifying spermidine and its metabolites.
Liquid Chromatography-Mass Spectrometry (LC-MS): This combined method enhances the ability to analyze low-abundance compounds, such as spermidine, with exceptional accuracy.
Nuclear Magnetic Resonance (NMR) Spectroscopy: NMR provides structural information about spermidine and its interaction with biomolecules, contributing to a better understanding of its biological roles.
Sample Requirements for Spermidine Analysis
| Sample Type | Volume Required | Collection Method | Storage Conditions |
|---|---|---|---|
| Blood (plasma) | 1 mL | EDTA or heparin tubes | -80°C for long-term storage |
| Urine | 5 mL | Clean catch method | -20°C for up to 3 months |
| Tissue Samples | 50 mg | Fresh or frozen tissue | -80°C for long-term storage |
| Cell Cultures | 1 mL | Standard cell culture | Process immediately or store at -80°C |
Note: It is critical to avoid contamination and ensure proper handling during sample collection and storage.
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
This study investigates the role of polyamine metabolism in immune cell dysfunction, particularly in the oral mucosa of people living with HIV (PLWH). Chronic HIV infection is associated with systemic inflammation, immune dysregulation, and increased risk of comorbidities. By comparing the protein, transcriptome, and metabolome profiles of uninfected and HIV-positive individuals, the researchers found elevated polyamine synthesis during HIV infection, which contributes to the dysfunction of regulatory T cells (Treg) and Th17 cells. Key enzymes, such as ornithine decarboxylase-1 (ODC-1), drive the production of polyamines like putrescine, spermidine, and spermine, which further impact T cell function and contribute to oral mucosal inflammation. The study provides insights into how polyamine metabolism contributes to immune dysfunction in chronic viral infections like HIV.
Materials & Methods
Human Samples
Human samples, including gingival biopsies and saliva, were collected from healthy individuals and those living with HIV (PLWH) with informed consent under a University Hospitals Cleveland Medical Center-approved protocol. Participant characteristics are detailed in Supplementary Table 1. Fresh gingival biopsies were processed for flow cytometry, and discarded palatine tonsils were obtained from tonsillectomy surgeries. A single-cell suspension of tissues was prepared using Collagenase 1A digestion and Ficoll-Paque PLUS centrifugation.
HTOC Cultures and HIV Infection
HTOC cultures were established by resuspending tonsillar cells at 1 million cells per well in transwell plates, stimulated with TCR activating antibodies and cytokines for 24–36 hours, and then infected with HIV X4-tropic NL43-GFP-IRES-Nef or HIV-NLGNef viruses. After 24–36 hours, Efavirenz (ARI) was added, and cells were expanded for 4–7 days before assays.
Cell Culture Reagents and Inhibitors
TCR stimulating antibodies were obtained from BD Biosciences and Thermofisher Scientific. Recombinant TGF-β1 and IL-2 were sourced from R&D Systems and BioLegend. Other reagents, including cytokines and inhibitors, were from various vendors. CD4+ T cells were purified, and lentiviral transduction was used to introduce control or ODC-1 shRNA.
Flow Cytometry
Flow cytometry employed fluorochrome-conjugated antibodies for various markers, including ROR-γt and HIF-1α. Cells were stained and analyzed on BD Fortessa cytometers, with specific gating to identify lymphocytes and CD4+ or CD8− populations.
Polyamine Assay
Polyamine levels in cell lysates and supernatants were measured using a fluorimetric method from BioVision. Cells were lysed, treated with a cleanup mix, and analyzed for polyamine content, normalizing concentrations to viable cell numbers.
Salivary Metabolome Analysis
Saliva samples were stored at −80 °C. Metabolite extraction for LC-MS involved vortexing with 80% methanol, centrifugation, and preparation for analysis. Q Exactive MS was used for separation, with a mobile phase of formic acid-water and acetonitrile.
RNA Sequencing and Metabolome Data Analysis
Gingival cells were enriched, and RNA was extracted for sequencing using the NEB Next® Ultra™ RNA Library Prep Kit. Sequencing was performed on HiSeq2500, followed by bioinformatic analysis to normalize gene reads and compare expression levels between controls and HIV+ individuals.
Targeted Polyamine Quantification Using LC-MS
Polyamine quantification involved standard solutions and cellular lysates, reacting with dansyl chloride. UPLC-MRM/MS analysis was conducted on an Agilent UHPLC system, normalizing data to input cells.
Statistical Analyses
Statistical significance was determined with *P < 0.05, using GraphPad Prism 8 for calculations. Mann–Whitney tests and one- or two-way ANOVA were employed for multiple comparisons, with Bonferroni t-tests as post hoc tests.
Results
Effect of Polyamines and EIF5A Hypusination on TregDys/Th17 Ratios:
Polyamine enrichment and EIF5A hypusination were investigated for their role in HIV-mediated increase in TregDys/Th17 ratios.
The addition of exogenous polyamines (putrescine, spermidine) or the DHPS inhibitor GC7 mimicked some effects of HIV infection, specifically increasing TregDys frequencies without restoring Th17 depletion.
Exogenous polyamines significantly elevated TregDys/Th17 ratios by increasing TregDys frequencies, though the increase was not as pronounced as in HIV-infected cultures.
ODC-1 inhibition, GC7 treatment, and exogenous spermidine reduced non-Treg cell viability more than Treg or TregDys cells, without inducing autophagic cell death.
Polyamines Induce TregDys Cells and EIF5A Hypusination:
Polyamines enhanced EIF5A expression and hypusination while downregulating ODC-1 in CD4+ T cells.
Polyamines promoted the expansion of FOXP3+ PD-1+ IFN-γ+ TregDys cells without inducing general IFN-γ expression in non-Treg CD4+ cells.
Spermidine further downregulated IFN-γ in uninfected non-Treg CD4+ cells.
FOXP3 expression was induced in Th1-like cells (PD-1+ IFN-γ+), contributing to TregDys and Th cell infidelity during HIV infection.
Polyamines upregulated Amphiregulin (AREG) and increased KI-67 expression, indicating TregDys proliferation.
HIV-1 infection increases polyamines in TCR-activated HTOC CD4+ T cells, dependent on ODC-1 activity. Following HIV infection and treatment with ARI and ODC-1 inhibitor I, polyamine levels (putrescine, spermidine, spermine, cadaverine) were quantified relative to uninfected controls. Data represent means and standard deviations from three independent experiments (n = 3), with significant differences determined by one-tailed t-tests (*P = 0.0126–0.0243; **P = 0.0038–0.0085). Absolute polyamine levels from one experiment are also shown. Thermospermine was not detected.
Correlation of TregDys/Th17 Ratios with Polyamine Levels in HIV+ Patients:
HIV+ patients showed a significant reduction in Th17 cells (~11%) in oral mucosa compared to healthy controls (~25%). Increased TregDys/Th17 ratios were found in the oral mucosa of HIV+ patients, similar to the results observed in vitro. Putrescine levels in saliva correlated with elevated TregDys/Th17 ratios and immune hyperactivation in the oral mucosa. These findings linked aberrant polyamine pathways to persistent immune dysregulation in chronic HIV infection.
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.
How is spermidine extracted from biological samples for analysis?
Spermidine extraction is a critical step that requires precise techniques to ensure accurate quantification. The extraction method typically involves the use of organic solvents such as perchloric acid or methanol, which help break down cellular components and isolate spermidine from other metabolites. This process often includes a protein precipitation step to remove unwanted proteins, followed by centrifugation to separate the supernatant containing spermidine. Depending on the biological matrix (e.g., blood, urine, tissue), the extraction process may be tailored to optimize recovery and ensure that no significant loss of spermidine occurs. After extraction, the samples are usually purified and concentrated to improve detection sensitivity during analysis.
What factors can affect spermidine stability during sample collection and storage?
Spermidine, like many small molecules, is sensitive to environmental conditions, including temperature, pH, and light exposure. To maintain its stability, it is crucial to store samples at very low temperatures (typically -80°C for long-term storage) to prevent degradation. Improper handling, such as repeated freeze-thaw cycles or exposure to room temperature for extended periods, can lead to spermidine degradation or conversion to other metabolites, affecting the accuracy of analysis. Additionally, using proper collection tubes (e.g., EDTA or heparin for blood samples) can prevent enzymatic activity that may alter spermidine levels.
Can spermidine levels vary significantly between different biological matrices?
Yes, spermidine levels can vary widely depending on the biological matrix being analyzed. For example, tissues like the liver and brain generally have higher concentrations of spermidine compared to fluids such as blood or urine. Additionally, spermidine levels can fluctuate due to physiological factors, such as age, diet, or health conditions, which may affect its synthesis and degradation in different tissues. This makes it important to select the appropriate matrix for your study, depending on the research question or target pathway. Our team at Creative Proteomics can provide guidance on which biological matrices are most suitable for your specific research objectives.
What challenges are associated with spermidine quantification in low-abundance samples?
Quantifying spermidine in low-abundance samples, such as those from specific cell types or rare tissues, presents technical challenges due to the compound's low concentration. However, using highly sensitive techniques like Liquid Chromatography-Mass Spectrometry (LC-MS), we can detect spermidine even at trace levels. Sample preparation steps, such as concentration and purification, are also crucial to enrich spermidine from the sample and reduce matrix interference. For ultra-low concentrations, additional optimization, such as using isotopic internal standards, can improve accuracy and consistency in quantification.
How do you ensure the accuracy and reproducibility of spermidine analysis?
Accuracy and reproducibility are ensured through rigorous calibration, the use of internal standards, and consistent methodological protocols. At Creative Proteomics, we employ validated analytical methods with calibration curves that span the expected concentration range of spermidine in your samples. Internal standards (often isotopically labeled spermidine) are added to every sample to account for any variability in extraction, handling, or instrument sensitivity. Additionally, all analyses are performed in duplicate or triplicate, and we regularly run quality control (QC) samples to monitor instrument performance and method consistency across different batches.
Can I analyze other polyamines alongside spermidine in the same sample?
Yes, our platforms allow for the simultaneous analysis of other polyamines, such as putrescine and spermine, alongside spermidine. This is particularly useful for researchers studying polyamine metabolism, as these molecules often exist in tightly regulated pathways and influence each other's biological roles. We utilize advanced methods like LC-MS, which can distinguish and quantify multiple polyamines in a single run, providing a comprehensive profile of polyamine levels in your sample. This approach allows for a more complete understanding of the polyamine pathway and its impact on biological functions.
What sample volume is required for spermidine analysis, and can you handle limited sample quantities?
The volume required for spermidine analysis depends on the biological matrix and the sensitivity of the detection method being used. Generally, we recommend 1 mL of plasma or 5 mL of urine, but for tissues, 50 mg is often sufficient. However, if you are working with limited sample quantities, such as small biopsies or rare cell populations, we can still accommodate your needs by optimizing our extraction and detection protocols for smaller volumes. We have specialized workflows designed to maximize data output from minimal sample amounts without compromising accuracy or reproducibility.
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: https://doi.org/10.1038/s41467-023-36163-2
Supplementation of spermidine at 40 mg/day has minimal effects on circulating polyamines: an exploratory double-blind randomized controlled trial in older men.
Keohane, Patrick, et al.
Journal: Nutrition Research
Year: 2024
DOI: https://doi.org/10.1016/j.nutres.2024.09.012
The Suppression of the KRAS G12D-Nrf2 Axis Shifts Arginine into the Phosphocreatine Energy System in Pancreatic Cancer Cells.
Di Giorgio, Eros, et al.
Journal: Cell Chemical Biology
Year: 2023
DOI: http://dx.doi.org/10.2139/ssrn.4318051
Disruption of CYCLOPHILIN 38 function reveals a photosynthesis-dependent systemic signal controlling lateral root emergence.
Duan, Lina, et al.
Journal: bioRxiv
Year: 2020
DOI: https://doi.org/10.1101/2020.03.11.985820
