Pentose Phosphate Pathway Analysis Service

What is Pentose Phosphate Pathway?

The Pentose Phosphate Pathway (PPP) is a metabolic pathway parallel to glycolysis that plays a crucial role in cellular metabolism. It primarily functions to generate NADPH and ribose-5-phosphate. NADPH is essential for biosynthetic reactions and antioxidant defense, while ribose-5-phosphate is a key component in nucleotide synthesis. The PPP consists of two main phases: the oxidative phase, where glucose-6-phosphate is oxidized to produce NADPH and ribulose-5-phosphate, and the non-oxidative phase, which converts ribulose-5-phosphate into ribose-5-phosphate and other sugars. This pathway is vital for cellular functions including anabolic reactions, cellular redox balance, and maintaining metabolic flexibility.

LC-MS/MS platform enable simultaneous relative and absolute quantification of multiple metabolites in a biological system. The results are widely used for biomarker discovery by investigating the relative changes in metabolite concentrations. The resulting data have been widely used to for biomarker discovery. By providing the exact molecular weights and retention time, LC-MS/MS techniques serves as a powerful analytical tool for identification and quantification of small molecules (metabolites). Creative Proteomics has established sensitive, reliable, and accurate HPLC-MS/MS method for quantification targeted metabolites in pentose phosphate pathway metabolites.

Pentose Phosphate Pathway Analysis at Creative Proteomics

Comprehensive Metabolic Profiling

Our metabolic profiling services deliver a thorough analysis of key PPP metabolites, such as glucose-6-phosphate, 6-phosphogluconate, and ribulose-5-phosphate. By measuring these metabolites with high precision, we help you assess the pathway's activity and its impact on cellular functions.

Flux Analysis and Pathway Modulation

Using advanced flux analysis techniques, we track the flow of metabolites through the PPP. By employing stable isotope-labeled substrates, we provide detailed insights into metabolic fluxes, helping you understand how the pathway adapts under different conditions or in various disease states.

List of Pentose Phosphate Pathway Metabolites We Can Analyze

Analytical Techniques for Pentose Phosphate Pathway Analysis

Mass Spectrometry-Based Metabolomics

Mass spectrometry (MS) is a cornerstone of our analytical approach. We utilize high-resolution instruments, such as the Orbitrap and Q-TOF (Quadrupole Time-of-Flight) mass spectrometers, to analyze PPP metabolites with exceptional sensitivity and specificity. These advanced MS systems allow for precise identification and quantification of metabolites like glucose-6-phosphate, 6-phosphogluconate, and ribulose-5-phosphate, providing crucial data on their concentrations and metabolic fluxes.

High-Performance Liquid Chromatography (HPLC)

Our HPLC systems, including those equipped with UV/Vis detectors and mass spectrometry couplings, are instrumental in separating and quantifying PPP intermediates. We employ various HPLC techniques, such as reverse-phase chromatography and ion-exchange chromatography, to isolate specific metabolites from complex biological samples. Instruments like the Agilent 1260 Infinity and Waters Acquity UPLC systems ensure high resolution and accuracy in our analyses.

Enzyme Activity Assays

To assess enzyme activity within the PPP, Creative Proteomics uses advanced spectrophotometric and fluorometric assays. Our equipment includes the BioTek Synergy and Molecular Devices SpectraMax plate readers, which measure the rate of substrate conversion by key PPP enzymes such as glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD). These assays provide valuable information on enzyme kinetics and regulatory mechanisms.

Sample Requirements for entose Phosphate Pathway Analysis

Report

• A detailed technical report will be provided at the end of the whole project, including the experiment procedure, MS/MS 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.

Lipin-1 regulates lipid catabolism in pro-resolving macrophages.

Journal: bioRxiv

Published: 2020

Background

Macrophages, essential for immune response and tissue repair, polarize into pro-inflammatory (M1) and pro-resolving (M2) phenotypes with distinct metabolic profiles. M1 macrophages rely on glycolysis and lipid synthesis, while M2 macrophages depend on oxidative metabolism and β-oxidation for energy. Lipin-1, a key regulator of lipid metabolism, plays dual roles: its enzymatic activity promotes inflammation, while its nuclear role supports tissue repair. Understanding how lipin-1 influences macrophage polarization and β-oxidation is crucial, as these mechanisms are linked to diseases like atherosclerosis and liver disease.

Materials & Methods

Animal Models:

• Mice aged 8 to 10 weeks were used, with lipin-1 genetically modified to delete either its enzymatic activity or the entire protein in myeloid cells.
• Control mice were lipin-1^flox/flox littermates.

Generation of Bone Marrow-Derived Macrophages (BMDMs):

• BMDMs were cultured from the bone marrow of lipin-1 modified and control mice for 7 to 10 days.

Mitochondrial Bioenergetics:

• Oxygen consumption rates (OCR) were measured using the Seahorse XF24 extracellular flux analyzer.
• Macrophages were treated with mitochondrial function inhibitors to assess the impact of lipin-1 on energy production.

Targeted Metabolomic Profiling:

Cell pellets from macrophages were sent to Creative Proteomics for targeted metabolomic, focusing on central carbon metabolism pathways.

Lipid Uptake:

Lipid uptake was assessed using BODIPY-labeled palmitate and quantified by flow cytometry.

Statistical Analysis:

Data were analyzed using GraphPad Prism, with appropriate statistical tests applied for accurate result interpretation.

Results

Lipin-1 and Metabolic Activity:

Lipin-1 is essential for increased metabolic activity in lipid catabolic states. Enzymatic activity of lipin-1 was not necessary for IL-4-induced oxygen consumption, but its transcriptional coregulatory function was crucial. BMDMs from lipin-1 KO mice failed to increase OCR in response to IL-4, unlike WT and EKO mice.

Lipid Utilization:

Lipin-1 is critical for lipid breakdown and utilization. IL-4-stimulated WT macrophages showed increased OCR with palmitate, indicating β-oxidation. In contrast, lipin-1 KO macrophages did not show increased OCR with palmitate. Lipin-1 does not affect lipid uptake as indicated by similar palmitate uptake levels in both WT and KO macrophages.

Metabolic Pathways:

Lipin-1 KO macrophages exhibited altered metabolism with increased glycolytic metabolites and decreased TCA cycle intermediates. IL-4 stimulation further exacerbated these metabolic changes, indicating impaired oxidative metabolism. There was a significant decrease in NADH levels and an increase in NADPH levels in lipin-1 KO macrophages, suggesting disrupted central carbon metabolism.

Lipin-1 regulates macrophage metabolism.

Efferocytosis:

Lipin-1 is involved in efferocytosis, with lipin-1 KO macrophages showing reduced ability to clear apoptotic cells compared to WT. The transcriptional coregulatory function of lipin-1, rather than its enzymatic activity, promotes efferocytosis. Lipin-1 KO macrophages had a defect in efferocytosis despite normal MerTK expression levels.

Oxidative Metabolism During Efferocytosis:

Both WT and EKO macrophages increased OCR in response to apoptotic cells, but lipin-1 KO macrophages failed to do so. This indicates that the transcriptional coregulatory function of lipin-1 is necessary for β-oxidation of AC-derived lipids during efferocytosis.

Reference

1. Schilke, Robert M., et al. "Lipin-1 regulates lipid catabolism in pro-resolving macrophages." bioRxiv (2020): 2020-06.