Prostaglandins Analysis Service

What are Prostaglandins?

The prostaglandins (PG) are a set of physiologically active lipid compounds with diverse hormone-like effects in animals. Prostaglandins are derived enzymatically from fatty acids. A prostaglandin contains 20 carbon atoms, including a 5-carbon ring. They are a sub-family of eicosanoids and of the prostanoid class of fatty acid derivatives. Prostaglandins have two derivatives: prostacyclins and thromboxanes. Prostacyclins are powerful locally acting vasodilators and can inhibit the aggregation of blood platelets. Through their role in vasodilation, prostacyclins also play important roles in inflammation. They are often synthesized in the walls of blood vessels and serve the physiological function of preventing needless clot formation, as well as regulating the contraction of smooth muscle tissue. On the contrary, thromboxanes are vasoconstrictors and facilitate platelet aggregation. Their names come from their role in clot formation (thrombosis).

Prostaglandins have been found in almost every tissue and organin humans and other animals. They are produced by almost all nucleated cells. They are autocrine and paracrine lipid mediators that act upon endothelium, platelets, uterine and mast cells. They are usually synthesized in the cell from the essential fatty acids (EFAs). An intermediate arachidonic acid is produced from diacylglycerol via phospholipase-A2, then brought to either the lipoxygenase pathway orthe cyclooxygenase pathway. The lipoxygenase enzyme pathway is active in leukocytes and in macrophages and synthesizes leukotrienes. Alternatively, the cyclooxygenase pathway produces prostacyclin, thromboxane and prostaglandin D, E and F.

There are typically ten known prostaglandin receptors on diverse cell types. Prostaglandins ligate a subclass of cell surface seven-transmembrane receptors, G-protein-coupled receptors (GPCRs). These receptors are termed as DP1-2, EP1-4, FP, IP1-2, and TP, corresponding to the receptors that ligate the corresponding prostaglandin.

These various receptors means that prostaglandins act on a large group of cells and have a wide variety of effects such ascausing constriction or dilation in vascular smooth muscle cells, causing aggregation or disaggregation of platelets, sensitizing spinal neurons to pain, inducing labor, decreasing intraocular pressure, regulating inflammation, regulating calcium movement, regulating hormones, and controlling cell growth, etc.

Prostaglandins Analysis in Creative Proteomics

At Creative Proteomics, we offer metabolomics services, including prostaglandin analysis, designed to meet the diverse needs of our clients in both research and clinical settings. Our services are characterized by their precision, reliability, and customization to ensure that we deliver the highest quality results.

Quantitative Analysis of Prostaglandins

Quantitative analysis is essential for measuring the exact concentration of prostaglandins in various biological samples. Our quantitative analysis service includes:

• Absolute Quantification: Using internal standards, we accurately measure the levels of specific prostaglandins.
• Relative Quantification: Comparing the levels of prostaglandins across different samples to identify changes or trends.

Qualitative Analysis of Prostaglandins

Qualitative analysis focuses on identifying the presence and types of prostaglandins in samples. Our services include:

• Identification of Prostaglandin Species: Detecting different types of prostaglandins present in the sample.
• Structural Characterization: Detailed analysis of the molecular structure of prostaglandins to understand their specific roles and functions.

Metabolite Profiling

Metabolite profiling involves the comprehensive analysis of prostaglandin metabolites, providing insights into metabolic pathways and biological processes. Our metabolite profiling service includes:

• Pathway Analysis: Mapping the metabolic pathways involving prostaglandins to understand their biological roles.
• Biomarker Discovery: Identifying potential biomarkers for disease diagnosis and therapeutic targets.

Custom Analytical Solutions

We understand that every research project has unique requirements. Our custom analytical solutions are tailored to meet specific client needs, including:

• Custom Method Development: Developing and validating custom analytical methods for specific prostaglandins or sample types.
• Specialized Sample Preparation: Tailoring sample preparation techniques to ensure optimal analysis of prostaglandins in various matrices.

Data Analysis and Interpretation

Interpreting the data obtained from prostaglandins analysis is crucial for deriving meaningful insights. Our data analysis and interpretation service includes:

• Statistical Analysis: Applying advanced statistical methods to analyze the data and identify significant findings.
• Bioinformatics Support: Providing bioinformatics tools and expertise to interpret complex data sets and generate actionable insights.

Analytical Techniques for Prostaglandins Analysis

High-Performance Liquid Chromatography (HPLC)

HPLC is a fundamental technique in prostaglandins analysis due to its high resolution, sensitivity, and versatility. At Creative Proteomics, we utilize the Agilent 1260 Infinity II HPLC System for:

• Separation of Prostaglandins: Efficiently separating different prostaglandin species within complex biological samples.
• Quantification: Accurate measurement of prostaglandin concentrations using calibrated standards.
• Detection Methods: Employing various detectors such as UV, fluorescence, and mass spectrometry (MS) to enhance sensitivity and specificity.

Liquid Chromatography-Mass Spectrometry (LC-MS)

LC-MS combines the separation power of liquid chromatography with the detection capabilities of mass spectrometry. Our Thermo Scientific Q Exactive Orbitrap LC-MS/MS system is highly effective for:

• High Sensitivity and Specificity: Detecting and quantifying prostaglandins at very low concentrations.
• Identification of Metabolites: Detailed profiling of prostaglandin metabolites and their biological pathways.
• Structural Elucidation: Providing molecular weight and structural information of prostaglandins and their derivatives.

Gas Chromatography-Mass Spectrometry (GC-MS)

GC-MS is ideal for analyzing volatile and thermally stable prostaglandins. At Creative Proteomics, we use the Agilent 7890B GC System coupled with 5977B MSD for:

• High Resolution: Excellent separation of prostaglandin compounds in complex mixtures.
• Quantitative Accuracy: Precise quantification of prostaglandins in various biological matrices.
• Volatile Compound Analysis: Effective for analyzing prostaglandins and their metabolites that can be vaporized without decomposition.

Tandem Mass Spectrometry (MS/MS)

MS/MS involves multiple stages of mass spectrometry, providing detailed structural information about prostaglandins. Our Thermo Scientific TSQ Altis Triple Quadrupole Mass Spectrometer is beneficial for:

• Structural Characterization: Offering comprehensive insights into the molecular structure of prostaglandins.
• Metabolite Identification: Identifying and characterizing prostaglandin metabolites with high accuracy.
• Enhanced Selectivity: Improving the selectivity and sensitivity of prostaglandin analysis through multiple fragmentation stages.

Ultra-Performance Liquid Chromatography (UPLC)

UPLC is an advanced form of liquid chromatography that provides faster and higher resolution separations. At Creative Proteomics, we use the Waters ACQUITY UPLC System for:

• Rapid Analysis: Significantly reducing analysis time while maintaining high resolution.
• High Throughput: Suitable for large-scale studies and high-throughput screening.
• Enhanced Sensitivity: Improving the detection limits of prostaglandins in various samples.

Sample Requirements for Prostaglandins Analysis

Important Considerations:

• Sample Collection: Ensure proper collection and handling to avoid contamination or degradation of prostaglandins.
• Storage Conditions: Store samples at -80°C if they are not analyzed immediately to preserve the integrity of prostaglandins.
• Avoid Repeated Freeze-Thaw Cycles: Minimize freeze-thaw cycles to prevent degradation of prostaglandins.
• Labeling and Documentation: Clearly label samples with relevant information, including sample type, collection date, and any pre-treatment details.

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 or ug/mg (tissue), and CV's are generally<10%.

Metabolomic profiling implicates mitochondrial and immune dysfunction in disease syndromes of the critically endangered black rhinoceros (Diceros bicornis).

Journal: Scientific reports

Published: 2023

Background

The study aimed to characterize the metabolome of black rhinoceroses (Diceros spp.) to provide foundational data on serum metabolites across different subspecies, sexes, and health phenotypes. This research is crucial for understanding metabolic variations in ex-situ managed rhinos and could aid in developing diagnostic biomarkers and health management strategies.

Materials & Methods

Study Population and Sample Collection

Fourteen Eastern black rhinos (Diceros b. michaeli) and sixteen Southern black rhinos (Diceros b. minor) from multiple institutions in the United States were included in this study. A total of 97 serum samples were collected longitudinally, approximately every three months, aiming for four samples per rhino within a year. Veterinary staff collected the samples, which were shipped to the Smithsonian's National Zoo & Conservation Biology Institute and stored at −80 °C until analysis.

Untargeted Metabolomic Analysis

Serum samples (0.1 mL aliquots) were sent to Creative Proteomics for untargeted metabolomic analysis using liquid chromatography coupled with mass spectrometry (LC–MS). Standardized protocols were followed for sample preparation and LC–MS analysis.

• Instrumentation: Analysis was performed using an Ultimate 3000LC coupled with the ThermoQ Exactive MS platform.
• Sample Processing: Samples underwent extraction and derivatization according to established protocols before analysis.

Data Acquisition and Analysis

• Metabolite Identification: Compound Discover 3.0 software (Thermo) was utilized for metabolite identification based on retention time and ion signals in both positive and negative ionization modes.
• Statistical Analysis: Data were processed using SIMCA-P 14.1 for visualization and outlier detection. Partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were employed to identify significant metabolites distinguishing between subspecies, sexes, and health phenotypes.
• Biomarker Discovery: Significant metabolites were identified based on VIP scores (VIP > 1.5) and t-tests (p < 0.05). Hierarchical clustering, principal component analysis (PCA), and volcano plots were used to visualize and interpret metabolic variations.

Quality Control

Quality control measures included the use of standard reference materials and replicate analyses to ensure data accuracy and reproducibility.

Results

Metabolic Profiles of Eastern and Southern Black Rhinos

Metabolomic analysis revealed distinct serum metabolic profiles between Eastern (Diceros b. michaeli) and Southern (Diceros b. minor) black rhinos. A total of 636 metabolites were consistently detected across all samples, with 352 identified in negative ion mode and 284 in positive ion mode.

Serum metabolome profile for eastern versus southern subspecies comparison relative to the eastern subspecies.

Serum metabolome profile for sex comparison relative to females.

Serum metabolome profile for health phenotype comparison (healthy vs. inflammatory) relative to inflammatory phenotype.

Subspecies Comparison

Differential Metabolite Composition: Principal Component Analysis (PCA) and hierarchical clustering highlighted significant differences in metabolite composition between Eastern and Southern black rhinos. Notably, metabolites involved in lipid metabolism, amino acid derivatives, and oxidative stress pathways showed distinct patterns between the two subspecies.

Specific Metabolites: Metabolites such as arachidonic acid derivatives and iron-related compounds were found to be significantly different between subspecies, suggesting potential metabolic adaptations or differences in physiological responses.

Sex-Based Differences

Sexual Dimorphism in Metabolites: Analysis revealed sex-specific metabolic differences within each subspecies. Male and female black rhinos exhibited distinct metabolic profiles, particularly in metabolites related to hormonal regulation, lipid metabolism, and antioxidant defense mechanisms.

Health Phenotype Analysis

Metabolic Variations in Health Phenotypes: Comparison between healthy and inflammatory phenotype cohorts showed significant alterations in metabolite profiles associated with immune function, oxidative stress, and inflammatory markers. Principal metabolites contributing to phenotype discrimination included cytokines, oxidative stress markers, and metabolic intermediates indicative of inflammatory responses.

Biomarker Potential

Candidate Biomarkers: Several metabolites emerged as potential biomarkers for health status differentiation among black rhinos, highlighting their utility in monitoring health and disease progression in managed populations.

Statistical Analysis

Statistical Significance: Statistical tests, including partial least squares discriminant analysis (PLS-DA) and t-tests, confirmed the robustness of identified biomarkers and their significance in differentiating between subspecies, sexes, and health phenotypes.

Receiver operating characteristic (ROC) curve-based approach for identifying potential biomarkers and evaluating their performance using area under the curve (AOC).

Reference

1. Corder, Molly L., et al. "Metabolomic profiling implicates mitochondrial and immune dysfunction in disease syndromes of the critically endangered black rhinoceros (Diceros bicornis)." Scientific Reports 13.1 (2023): 15464.