Ubiquinone Analysis Service

What is Ubiquinone?

Ubiquinones, also known as CoEnzyme Q (CoQ), are lipid-soluble molecules found in the mitochondria of cells. They play a crucial role in the electron transport chain, a series of reactions essential for cellular energy production. The most well-known ubiquinones are CoQ9 and CoQ10, with the number indicating the length of their isoprene side chains. Ubiquinones can exist in three oxidation states: fully oxidized (ubiquinone), partially reduced (semiquinone), and fully reduced (ubiquinol). These forms allow ubiquinones to function not only in electron transport but also as powerful antioxidants, protecting cells from oxidative damage.

Ubiquinone Analysis in Creative Proteomics

At Creative Proteomics, we offer a comprehensive range of ubiquinone analysis services designed to meet the diverse needs of researchers and industry professionals. Our suite of analytical projects leverages advanced technologies and methodologies to ensure precise, accurate, and reliable results.

Quantitative Analysis of CoQ10 and CoQ9

We specialize in the quantitative analysis of CoQ10 and CoQ9, employing HPLC-MS. This technique allows us to quantify these ubiquinones in various biological samples, which is crucial for assessing ubiquinone levels in health and disease studies, nutritional supplement efficacy, and metabolic profiling.

Ubiquinone Profiling

Our ubiquinone profiling service provides a comprehensive overview of all ubiquinone variants present in a sample. Utilizing UPLC coupled with MS, we can investigate the complete ubiquinone spectrum in tissues, cells, and other biological matrices. This service is particularly useful for understanding the diversity and function of ubiquinones in different biological contexts.

Redox State Analysis

We offer redox state analysis to determine the oxidation states of ubiquinones, distinguishing between ubiquinone (oxidized), ubiquinol (reduced), and semiquinone (partially reduced). Using HPLC-MS with redox-sensitive detection, this analysis is essential for studying oxidative stress, antioxidant capacity, and mitochondrial function.

Ubiquinone Biosynthesis Pathway Analysis

For researchers investigating the biosynthesis of ubiquinones, we provide specialized analysis of intermediates and enzymes involved in ubiquinone biosynthesis pathways. This targeted metabolomics approach uses HPLC-MS and GC-MS to support research on genetic disorders affecting ubiquinone biosynthesis and drug development targeting these pathways.

Tissue-Specific Ubiquinone Distribution

We conduct tissue-specific ubiquinone distribution studies to measure and compare ubiquinone concentrations across different tissues and organs. This service, utilizing both HPLC-MS and UPLC-MS, is valuable for understanding the tissue-specific roles of ubiquinones and the pharmacokinetics of ubiquinone supplements.

Mitochondrial Ubiquinone Analysis

Our mitochondrial ubiquinone analysis focuses on quantifying and profiling ubiquinones specifically in isolated mitochondria. This is achieved through HPLC-MS with mitochondrial isolation protocols, aiding research into mitochondrial function, bioenergetics, and mitochondrial diseases.

Plasma and Serum Ubiquinone Detection

We detect and quantify ubiquinones in plasma and serum samples using HPLC-MS. This application is essential for biomarker discovery, clinical studies on cardiovascular health, and antioxidant status assessment.

Impact of Supplements on Ubiquinone Levels

To evaluate the impact of dietary supplements on ubiquinone concentrations, we offer services that utilize both HPLC-MS and UPLC-MS. These analyses support nutritional research, efficacy studies of CoQ10 supplements, and clinical trials.

Ubiquinone Turnover Rate Measurement

We measure the rate of ubiquinone turnover and degradation in cells and tissues. Using isotope labeling combined with HPLC-MS, this service is crucial for studying metabolic turnover, aging, and response to oxidative stress.

High-Throughput Screening for Ubiquinone Levels

For large-scale studies, epidemiological research, and high-throughput drug screening, we provide high-throughput screening for ubiquinone levels. Our automated HPLC-MS and UPLC-MS systems allow for the rapid screening of large numbers of samples, ensuring efficient and accurate data collection.

Analytical Techniques for Ubiquinone Analysis

High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS): We employ the Agilent 1260 Infinity II HPLC System with the Thermo Scientific Q Exactive HF-X Mass Spectrometer. This setup enables precise quantification of CoQ10 and CoQ9, redox state analysis, and detection of ubiquinones in plasma and serum.

Ultra-Performance Liquid Chromatography (UPLC) Coupled with Mass Spectrometry (MS): Using the Waters ACQUITY UPLC System and Waters Xevo G2-XS QTof Mass Spectrometer, we achieve high resolution and sensitivity for ubiquinone profiling across biological matrices.

Gas Chromatography-Mass Spectrometry (GC-MS): Our analysis utilizes the Agilent 7890B GC System coupled with the Agilent 5977B GC/MSD System, ideal for studying ubiquinone biosynthesis pathways and metabolic intermediates.

Redox-Sensitive Detection Methods: Equipped with the Shimadzu Nexera X2 UHPLC System and redox-sensitive detectors, we analyze ubiquinone redox states to understand oxidative stress and mitochondrial function.

Isotope Labeling Techniques: The Thermo Scientific TSQ Altis Triple Quadrupole Mass Spectrometer is used for precise measurement of ubiquinone turnover rates and metabolic dynamics in cells and tissues.

Automated High-Throughput Systems: Our Tecan Freedom EVO Platform and Agilent 1290 Infinity II UPLC System facilitate rapid screening of ubiquinone levels in large-scale studies and clinical trials.

Sample Requirements for Ubiquinone Analysis

Notes:

• Sample Collection: Use sterile and non-contaminated equipment. For plasma/serum, use EDTA or heparin as anticoagulants.
• Homogenization: Ensure complete homogenization of tissue samples in cold PBS to prevent degradation.
• Storage: Store samples at -80°C or in liquid nitrogen to maintain stability and prevent degradation.
• Avoid Freeze-Thaw Cycles: Minimize freeze-thaw cycles to preserve sample integrity.

For specific requirements or additional instructions related to your samples, please contact our support team.

Report

• A detailed technical report will be provided at the end of the whole project, including the experiment procedure, instrument parameters.
• Analytes are reported as uM or ug/mg (tissue), and CV's are generally<10%.
• The name of the analytes, abbreviation, formula, molecular weight and CAS# would also be included in the report.

Anti-Inflammatory Activity of Black Soldier Fly Oil Associated with Modulation of TLR Signaling: A Metabolomic Approach

Journal: International Journal of Molecular Sciences

Published: 2023

Background

The study of succinic acid, a key intermediate in cellular metabolism, has garnered significant attention due to its diverse industrial applications and biological importance. Succinic acid is pivotal in various metabolic pathways, including the tricarboxylic acid cycle, and its production pathways have been extensively studied to enhance yields and sustainability. Despite its natural occurrence in organisms, engineered microbial systems offer promising avenues for cost-effective and environmentally friendly production. Understanding the metabolic regulation and genetic engineering strategies for succinic acid production remains critical for advancing biotechnological applications.

Materials & Methods

Materials

Black Soldier Fly Oil: Obtained from black soldier fly larvae (Hermetia illucens) through a mechanical extraction process.

Cell Culture:

• Cell Lines: Human monocytic cell line (THP-1) and human embryonic kidney cells (HEK293).
• Culture Medium: RPMI-1640 supplemented with fetal bovine serum (FBS) and antibiotics.
• Activation Agents: Lipopolysaccharide (LPS) for inducing inflammatory responses.

Animals:

• Species: Male BALB/c mice.
• Treatment: Administered black soldier fly oil orally or topically.

Methods

Cell Viability Assay:

• Purpose: Determine cytotoxic effects of black soldier fly oil.
• Technique: MTT assay or similar viability tests.

In Vitro Inflammatory Model:

• Stimulation: THP-1 cells treated with LPS to induce inflammation.
• Treatment: Cells treated with varying concentrations of black soldier fly oil.
• Analysis: Measure secretion of pro-inflammatory cytokines (TNF-α, IL-6) using ELISA.

Metabolomic Profiling:

• Sample Preparation: Cell lysates or tissue samples prepared using appropriate extraction methods.
• Instrumentation: Liquid chromatography-mass spectrometry (LC-MS) or gas chromatography-mass spectrometry (GC-MS).
• Data Analysis: Software tools for metabolite identification and quantification.

Animal Studies:

• Administration: Mice administered black soldier fly oil orally or topically.
• Inflammatory Assays: Measure cytokine levels in serum or tissue homogenates.
• Histological Analysis: Evaluate tissue samples for inflammation markers.

Statistical Analysis:

• Methods: ANOVA, t-tests, or non-parametric tests.
• Significance: Criteria set at p < 0.05 for statistical significance.

Results

Suppression of TLR4-Mediated Proinflammatory Cytokines by Modified BSFL Oil (MBSFL) and C12:0 in Macrophages

Modified BSFL oil (MBSFL) significantly reduced TNFα mRNA expression and protein secretion in LPS-stimulated THP-1 and J774A.1 macrophages. It also decreased IL-6 and IL-1β protein secretion without affecting their transcription levels.

Suppression of Toll-like receptor (TLR) 4-mediated proinflammatory cytokines by MBSFL and C12:0 in macrophages.

MBSFL and C12:0 Reciprocally Modulate TLR2-Mediated Proinflammatory Cytokines Expression and Secretion in Macrophages

MBSFL reduced TNFα secretion induced by Pam3 in both THP-1 and J774A.1 cells. It also decreased IL-6 and IL-1β secretion induced by Pam3 in THP-1 cells. In contrast, linoleic acid (LA) and stearic acid (SL) showed less consistent effects on cytokine secretion.

Differential Transcriptome Profile upon Treatment with MBSFL or SL in TLR4-Activated Macrophages

MBSFL and SL treatment influenced distinct gene expression profiles in LPS-stimulated THP-1 cells. MBSFL upregulated genes involved in PPAR signaling and metabolic regulation, while SL affected cholesterol biosynthesis pathways.

Transcriptome analysis of MBSFL and SL effects on TLR4-activated macrophages.

Effect of BSFL Oil on DSS-Induced Colitis in Mice

BSFL oil reduced body weight loss and disease activity index in DSS-induced colitis mice. It preserved colon length and reduced spleen weight compared to soybean oil and palm oil diets.

Histopathological Signs of Colitis in DSS-Treated Mice as a Function of Diet Composition

DSS treatment induced lymphocyte infiltration in colonic mucosa, unaffected by diet type. Neutrophil infiltration and crypt loss were consistent across all diet groups.

Enhanced IgA Production in Response to DSS Treatment with BSFL Oil

BSFL oil-fed mice exhibited earlier and higher levels of fecal IgA following DSS treatment compared to soybean oil- and palm oil-fed mice.

Conclusion

Based on experimental findings, black soldier fly oil exhibits potent anti-inflammatory effects, potentially modulating inflammation by suppressing TLR signaling pathways. This discovery provides a theoretical basis for black soldier fly oil as a promising resource for anti-inflammatory therapeutics.

Reference

1. Richter, Hadas, Ofer Gover, and Betty Schwartz. "Anti-inflammatory activity of black soldier fly oil associated with modulation of tlr signaling: A metabolomic approach." International Journal of Molecular Sciences 24.13 (2023): 10634.