Succinic Acids Analysis Service

What are Succinic Acids?

Succinic acid, chemically known as butanedioic acid, is a naturally occurring dicarboxylic acid with the molecular formula C4H6O4. It is widely distributed in both plant and animal tissues and plays a pivotal role in intermediary metabolism. Structurally, succinic acid appears as a colorless crystalline solid that is soluble in water. In biological systems, succinic acid is involved in key biochemical pathways, most notably in the citric acid cycle (also known as the Krebs cycle) where it serves as an intermediate. This cycle is fundamental to cellular respiration, facilitating the conversion of nutrients, such as carbohydrates, fats, and proteins, into energy-rich molecules like ATP (adenosine triphosphate). Succinic acid also acts as an electron donor in the electron transport chain, a series of redox reactions crucial for ATP production. Beyond its biological functions, succinic acid finds applications in various industries, including pharmaceuticals, food production, and agriculture, owing to its roles in chemical synthesis and its potential therapeutic properties.

Succinic Acids Analysis in Creative Proteomics

Succinic Acids Quantitative Analysis

Accurate measurement of succinic acid levels using LC-MS/MS. This method ensures precise quantification in food, beverage, pharmaceutical, and biological samples.

Succinic Acids Structural Characterization

Identification and characterization of succinic acid variants and derivatives present in different matrices. This involves detailed structural profiling using advanced analytical techniques to understand its chemical composition and stability.

Biochemical Pathway Studies

Investigation of succinic acid's role in biochemical pathways, particularly its involvement in the citric acid cycle and energy metabolism. This includes elucidating its biosynthesis mechanisms and regulatory enzymes in various biological systems.

Comparative Analysis

Comparison of succinic acid profiles across different organisms, strains, or environmental conditions. This comparative analysis helps identify unique metabolic signatures and adaptations related to succinic acid production.

Drug Interaction Studies

Examination of how succinic acid levels and metabolism are influenced by pharmaceutical agents. This includes assessing the impact of drugs on succinic acid synthesis pathways and metabolic flux in cellular models.

Customized Research Projects

Tailored analysis services to meet specific research objectives or industrial requirements related to succinic acid analysis. This includes method development, validation, and specialized analytical support.

Analytical Techniques for Succinic Acids Analysis

LC-MS/MS (Liquid Chromatography-Mass Spectrometry)

LC-MS/MS combines the separating power of liquid chromatography with the sensitive detection capability of mass spectrometry. It separates compounds based on their interaction with the chromatographic column and detects and quantifies them based on their mass-to-charge ratio.

• Quantification: LC-MS/MS provides accurate quantification of succinic acid in various matrices such as biological samples, food, and pharmaceuticals, with high sensitivity and specificity.
• Identification: It identifies succinic acid based on its unique mass spectrum, confirming its presence and purity in complex mixtures.
• Structural Elucidation: Fragmentation patterns obtained from MS/MS analysis elucidate the structure of succinic acid and its derivatives, aiding in structural characterization.

HPLC (High-Performance Liquid Chromatography)

HPLC separates compounds based on their interaction with a chromatographic column packed with stationary phase materials. It uses a high-pressure pump to push the sample mixture through the column.

• Separation: HPLC separates succinic acid from other components in a sample, providing high resolution and reproducibility.
• Detection: Coupled with detectors like UV or RID (Refractive Index Detector), HPLC detects succinic acid by measuring its response to light or changes in refractive index.
• Quantification: It quantifies succinic acid levels in different sample types, supporting routine analysis and quality control.

NMR Spectroscopy (Nuclear Magnetic Resonance)

NMR spectroscopy detects the magnetic properties of atomic nuclei in a magnetic field. It provides detailed information about molecular structure, dynamics, and chemical environment.

• Structural Confirmation: NMR spectroscopy confirms the chemical structure of succinic acid and identifies functional groups.
• Purity Analysis: It assesses the purity of succinic acid samples by analyzing spectral shifts and patterns.
• Quantitative Analysis: NMR can quantify succinic acid in solution, providing insights into its concentration and stability.

FTIR Spectroscopy (Fourier Transform Infrared Spectroscopy)

FTIR spectroscopy measures the absorption of infrared light by a sample, revealing molecular vibrational modes and providing information about functional groups.

• Functional Group Analysis: FTIR identifies specific functional groups present in succinic acid, aiding in structural characterization and quality assessment.
• Rapid Analysis: It offers rapid qualitative analysis of succinic acid samples, supporting routine quality control in manufacturing processes.

Sample Requirements for Succinic Acids Analysis

Notes:

• Biological Samples: Use sterile techniques for collection to prevent contamination. Store at appropriate temperatures (-80°C for long-term storage) to maintain stability.
• Food and Beverage: Ensure samples are representative of the batch. Protect from light and air during storage and shipment.
• Pharmaceutical Formulations: Preserve sample integrity by avoiding exposure to light, moisture, and temperature extremes.
• Environmental Samples: Provide sufficient information on sample type (e.g., freshwater, agricultural soil) for accurate analysis.

For specific guidance on sample preparation or additional requirements, feel free to contact us.

Report

• A full report including all raw data, MS/MS instrument parameters and step-by-step calculations will be provided (Excel and PDF formats).
• Analytes are reported as uM, with CV's generally ~10%.

Vibrio cholerae Infection Induces Strain-Specific Modulation of the Zebrafish Intestinal Microbiome

Journal: Infection and Immunity

Published: 2021

Background

The study investigates the impact of various Vibrio cholerae strains on the intestinal microbiota of zebrafish, focusing on colonization patterns and associated changes in microbial diversity and metabolites. Zebrafish serve as a model organism due to their genetic tractability and physiological relevance to human intestinal microbiota studies. The study aims to elucidate how V. cholerae infections alter the zebrafish gut microbiome, potentially influencing host health and disease.

Materials & Methods

Bacterial Strains and Culture Conditions

Five V. cholerae strains (254-93, AM-19226, V52, E7946, N16961) were cultured in LB broth with streptomycin at 37°C. Culture densities were adjusted prior to zebrafish exposure.

Zebrafish Husbandry and Experimental Design

Wild-type AB zebrafish were housed under controlled conditions and fasted before experimentation. Fish were exposed to V. cholerae via immersion in sterile infection water, with control groups exposed to 1× PBS.

Intestinal Colonization Assessment

After exposure, zebrafish intestines were homogenized, and bacterial colonies were quantified on selective media. Tank water was also sampled and processed similarly.

DNA Extraction and Sequencing

DNA was extracted from zebrafish intestines and water samples for 16S rRNA gene sequencing using Illumina MiSeq. Data were processed using DADA2 to identify amplicon sequence variants (ASVs).

Diversity Analysis

Alpha (Chao1, Shannon-Wiener, inverse Simpson indices) and beta (Jaccard, Bray-Curtis dissimilarity) diversity indices were calculated. PCoA and PERMANOVA were used to assess microbiome variation among samples.

Metabolomics and Functional Analysis

Untargeted metabolomics of zebrafish intestinal homogenates was conducted using UPLC-TOF-MS. Sample preparation involved extraction with 80% methanol. The analysis focused on identifying metabolites such as succinic acids and their relative abundances in zebrafish intestinal samples.

Statistical Analysis

Data were analyzed using various statistical tests, including ANOVA and Tukey's test, to assess significance between experimental groups.

Results

Intestinal Colonization by V. cholerae

Intestinal colonization by V. cholerae strains was assessed through bacterial enumeration in zebrafish intestines. Significant differences were observed in colonization levels among the different strains (V. cholerae 254-93, AM-19226, V52, E7946, N16961). Strain E7946 exhibited the highest colonization levels, followed by strains AM-19226 and V52, whereas strains 254-93 and N16961 showed lower colonization efficiency.

Microbiome Diversity Analysis

• Alpha Diversity

Alpha diversity indices (Chao1, Shannon-Wiener, inverse Simpson) were calculated to assess species richness and evenness in zebrafish intestines following V. cholerae infection. Compared to controls, infected groups exhibited altered alpha diversity metrics, with significant differences observed in richness and evenness indices.

• Beta Diversity

Beta diversity analysis using PCoA based on Bray-Curtis dissimilarity matrices revealed distinct clustering patterns among V. cholerae-infected and control group. PERMANOVA analysis confirmed significant differences in microbiome composition between infection groups and controls.

β-Diversity of the zebrafish intestinal microbiome following V. cholerae 254-93 infection in a zebrafish host.

β-Diversity of the zebrafish intestinal microbiome following V. cholerae V52 infection in a zebrafish host.

Metabolomics Analysis

Untargeted metabolomics identified differential metabolic profiles in zebrafish intestines following V. cholerae infection. Metabolites associated with immune response and gut health were significantly altered in infected groups compared to controls.

Comparison of zebrafish intestinal microbiome β-diversities following V. cholerae infections. (A) β-Diversities of the zebrafish intestinal microbiome following a 24-hour V. cholerae infection expressed as PCoA plots based on Jaccard and Bray-Curtis dissimilarity indices. (B) P values indicating statistical differences of the PCoA plots shown in A.

Secreted Mucin Quantification

The Microtiter PAS assay quantified mucin levels in infection water, a biomarker for diarrhea. Significant increases in mucin secretion were observed in V. cholerae-infected groups compared to controls, correlating with infection severity.

Conclusion

V. cholerae infections in zebrafish induce substantial alterations in gut microbiota composition and metabolic profiles. These findings underscore the role of V. cholerae in reshaping host-associated microbial communities, potentially influencing disease susceptibility and host health. Further research into specific microbial interactions and metabolite pathways may elucidate mechanisms underlying V. cholerae pathogenicity and host-microbiome interactions, offering insights into therapeutic interventions for cholera and related gastrointestinal diseases.

Reference

1. Breen, Paul, et al. "Vibrio cholerae infection induces strain-specific modulation of the zebrafish intestinal microbiome." Infection and Immunity 89.9 (2021).