Spermine Analysis Service

What is Spermine?

Spermine is a naturally occurring polyamine that plays a critical role in cellular metabolism across virtually all living organisms. Its discovery dates back to 1678 when Antonie van Leeuwenhoek first identified it in human seminal plasma. Later, in 1888, German chemists Ladenburg and Abel introduced the name "spermin." The accurate chemical structure of spermine was finally established in 1926 by Dudley, Rosenheim, Starling, and Wrede.

Spermine exists predominantly as a polycation at physiological pH and is often found associated with nucleic acids. Its primary biological function includes stabilizing the helical structures of nucleic acids, notably in viruses. Furthermore, spermine and its related polyamines, such as spermidine and putrescine, are essential for several biochemical processes. These include the regulation of transcription and translation, modulation of ion channels, and the maintenance of cellular membrane integrity.

In mammals, spermine is synthesized from the precursor ornithine, which is critical for cellular growth. While spermine is abundant in various tissues, its levels are carefully regulated due to its role in essential processes such as cell cycle progression, protection against oxidative damage, and the stabilization of DNA structures.

Creative Proteomics offers targeted metabolomics analysis services to facilitate qualitative and quantitative analysis of spermine. Our services cater to researchers from various fields including molecular biology, biochemistry, and cellular biology, offering precise and reproducible spermine measurements.

Spermine Analysis in Creative Proteomics

Comprehensive Spermine Quantification

We provide accurate spermine quantification using a variety of cutting-edge analytical methods. These methods allow us to measure spermine in complex biological samples, such as serum, tissues, cell cultures, and biological fluids. Our multi-platform approach ensures high sensitivity, enabling us to detect spermine even at low concentrations.

Flexible Analysis Options: In Vitro and In Vivo Studies

Our services support both in vitro and in vivo studies, making it possible to explore spermine's role in different biological settings. Whether you're examining the effects of spermine on cultured cells or investigating its role in tissue-specific processes, our analyses are designed to accommodate your experimental setup.

Polyamine and Biogenic Amine Profiling

In addition to spermine, we offer full profiling of other related polyamines such as spermidine, putrescine, and additional biogenic amines. This comprehensive profiling allows researchers to gain a deeper understanding of the polyamine metabolic pathways, which are critical for processes such as cell growth, differentiation, and stress responses.

Tailored Analysis Solutions

Recognizing the unique requirements of each research project, we offer customized analysis services. Whether you're studying the regulatory effects of spermine on specific cellular functions, or its involvement in disease mechanisms, our flexible platform ensures that the analytical approach is adapted to your project's goals.

Detailed Reporting and Expert Consultation

We provide complete, high-quality reports with all data, including chromatograms, quantitative results, and detailed method descriptions. Our experts are also available for consultation and data interpretation, providing insights into the biological significance of the findings and helping you navigate through the complexities of polyamine research.

Analytical Techniques for Spermine Analysis

High-Performance Liquid Chromatography (HPLC)

HPLC is a primary method for spermine quantification, providing high resolution and precision. Using precolumn derivatization with DNS-Cl, we enhance detection sensitivity, making it ideal for analyzing spermine in complex biological matrices such as serum and tissues.

Instruments: Agilent 1260 Infinity II HPLC, Shimadzu Prominence HPLC

Liquid Chromatography-Mass Spectrometry (LC-MS)

LC-MS combines the separation capabilities of liquid chromatography with the sensitive detection of mass spectrometry. This technique provides both quantitative data and molecular structure information

Instruments: Thermo Fisher Q Exactive™, Agilent 6545 LC/Q-TOF

Gas Chromatography-Mass Spectrometry (GC-MS)

GC-MS is used for analyzing volatile or semi-volatile spermine derivatives, offering high sensitivity and specificity. It is especially suitable for specialized studies requiring detailed profiling of polyamines in complex samples.

Instruments: Agilent 7890B GC coupled with 5977B MS

Spectrophotometry-Based Methods

Spectrophotometric methods provide quick, cost-effective spermine quantification, suitable for high-throughput screening or preliminary studies where rapid results are needed.

Instruments: Thermo Fisher NanoDrop™ 2000, BioTek Synergy HTX

Sample Requirements for Spermine Analysis

Disruption of CYCLOPHILIN 38 function reveals a photosynthesis-dependent systemic signal controlling lateral root emergence.

Journal: bioRxiv

Published: 2020

Background

Polyamines, including putrescine, spermidine, and spermine, are vital for numerous biological processes like transcription, translation, cell growth, and aging. These molecules are fully protonated at physiological pH and predominantly associate with RNA and ribosomes to facilitate global protein synthesis. Polyamine regulation occurs through various mechanisms, such as gut absorption, de novo synthesis, and salvage pathways.

In the kidney, which is highly metabolically active, polyamine homeostasis is crucial and its disruption can lead to various diseases. Recent studies have identified altered polyamine metabolism as a common feature in multiple kidney injury models, with polyamines playing diverse roles, from protection to tissue damage. Understanding how polyamine regulation occurs, particularly through mechanisms like AZIN1 A-to-I RNA editing, is essential in defining the timeline and stages of kidney disease. The study of AZIN1 editing offers potential as a molecular clock to track disease progression in both kidney injuries and other conditions.

Materials & Methods

Malaria Cohort: RNA-seq data (GEO GSE52166) from whole blood samples before and after Plasmodium falciparum infection.

Human Kidney Biopsy: Bulk RNA-seq data from GSE139061 and Kidney Precision Medicine Project Atlas. Tissues processed with SMARTer Stranded Total RNA-Seq Kit v2 and sequenced on NovaSeq.

Generation of AZIN1 Cell Lines: CRISPR/Cas9 was used to generate HEK293T cell lines with specific AZIN1 edits. Cells were nucleofected and clones isolated.

Animal Models:

• Mice: C57BL/6J and AZIN1-edited mice used for kidney injury studies.
• Procedures: Endotoxin injection and ischemia-reperfusion injury performed.

Cells: HEK293T and AZIN1-edited cells cultured in DMEM with FBS and antibiotics.

dsRNA Analysis:

• UV Crosslinking: Total RNA extracted from mouse kidneys, digested with RNase A and RNase III, and analyzed for dsRNA.
• Immunoprecipitation: dsRNA isolated using anti-dsRNA antibody J2, RNA extracted and sequenced.

A-to-I Editing Analysis: Sequencing data analyzed using reditools2 and edgeR. Sites filtered by editing rate and sample coverage.

Nanopore RNA Sequencing: Direct cDNA sequencing of polyA+ mRNA from mouse kidneys.

AZIN1 Immunoprecipitation and Mass Spectrometry: FLAG-tagged proteins isolated and analyzed by LC-MS/MS.

Polyribosomal Profiling: Tissues and cells subjected to cycloheximide treatment, lysed, and analyzed on sucrose gradients.

Metabolomics:

• Untargeted Analysis: Performed at Metabolon Inc.
• Targeted Analysis: Includes measurement of spermine using dansyl chloride derivatization and UPLC-MS/MS.
• NAD+ Measurement: HPLC with UV detection.

Seahorse Bioenergetics Assays: Assessed cellular glycolysis and energy metabolism.

Real-time Cell Growth Monitoring: Used IncuCyte for live-cell imaging and growth quantification.

PCR: RNA extraction and reverse transcription followed by conventional PCR.

Western Blotting: Proteins extracted, separated by gel electrophoresis, and analyzed for various markers using specific antibodies.

Results

Polyamine Metabolism Changes

In the endotoxemia model, significant shifts in polyamine metabolism were observed. Early after endotoxin exposure, levels of spermidine and spermine increased, while putrescine levels decreased. This initial increase in polyamines suggests their role in the early inflammatory response. However, beyond 16 hours post-exposure, there was a marked depletion of polyamines, which correlated with decreased expression of key enzymes involved in polyamine synthesis, such as Ornithine Decarboxylase 1. This depletion aligns with the transition to a later stage of sepsis characterized by translation shutdown.

Impact of AZIN1 A-to-I Editing on Metabolism

AZIN1 A-to-I editing was found to influence polyamine metabolism. In CRISPR knock-in cell lines with A-to-I locked AZIN1, cells showed accelerated growth and enhanced metabolic flexibility compared to uneditable cells, especially under stress conditions such as nutrient deprivation. The A-to-I locked state maintained higher polyamine levels and exhibited better growth in response to urea supplementation, indicating a maximized polyamine synthesis.

In Vivo Metabolic Effects

In AZIN1 A-to-I edited mouse models, metabolic analysis revealed increased levels of NAD+ and polyamine-related metabolites, such as S-adenosylmethionine, following ischemia-reperfusion injury. These metabolites are crucial for maintaining cellular functions and metabolic flexibility. Elevated chiro-inositol levels were also observed, which may contribute to resilience against metabolic stress by enhancing glycolytic capacity.

Azin1 A-to-I locked state limits kidney injury by upregulating polyamines and other protective pathways.
(A) Sanger sequencing of wild-type, Azin1 A-to-I uneditable, and Azin1 A-to-I locked mice. (B) Serum creatinine levels 24 hours after ischemia-reperfusion injury in Azin1 A-to-I locked vs. uneditable mice. (C) Polyribosome profiling of kidneys from Azin1 A-to-I locked and uneditable mice, showing polysome-to-monosome ratios. (D) Western blot of hypusine levels in kidneys post-ischemia-reperfusion injury. (E) Volcano plot of top differentially expressed metabolites between Azin1 A-to-I locked and uneditable mice. (F) Heatmap of differentially expressed metabolites in Azin1 A-to-I locked vs. uneditable mice after ischemia-reperfusion. (G) Pathway enrichment analysis of metabolites in Azin1 A-to-I locked vs. uneditable kidneys. (H) Metabolite ratios mapped to the polyamine pathway. (I) Metabolite ratios mapped to the NAD+ biosynthesis pathway.

Reference

1. Heruye, Segewkal, et al. "Inflammation primes the kidney for recovery by activating AZIN1 A-to-I editing." bioRxiv (2023).