Pentose Phosphate Pathway Analysis Service

Also known as phosphogluconate pathway or the hexose monophosphate shunt, the pentose phosphate pathway (PPP) branches from glycolysis at the first committed step of glucose metabolism. With glucose-6-phosphate as a primary substrate, this pathway is catalyzed by hexokinase and generates building blocks for ribonucleotides synthesis and NADPH, which is required for the clearance of reactive oxygen species and fatty acids synthesis. It is shown in some report that pentose phosphate pathway plays a key role in assisting glycolytic cancer cells to combat against oxidative stress and meet their anabolic demands.

Because pentose phosphate pathway is related to the hemolytic anemia induced by oxidant agents, Fava beans and certain drugs like antimalarial drugs, it gained extensive attention about 90 years ago. Then it is discovered that people susceptible to hemolytic anemia are defect in the gene response for the crucial enzyme catalyzing the first committed step in the PPP, glucose-6-phosphate dehydrogenase (G6PDH). Since pentose phosphate pathway is the exclusive source of NADPH, a major scavenger of reactive oxygen species (ROS), in red blood cells, the deficient PPP pathway makes red blood cells more vulnerable to oxidants.

The PPP comprises of two phases: the oxidative branch and the nonoxidative branch. The oxidative branch generates NADPH and ribonucleotides and is composed of three irreversible reactions. In the first reaction, catalyzed by G6PDH, glucose-6-phophate (G6P) dehydrogenates and form NADPH and 6-phosphogluconlactone, which in turn converts to 6-phosphogluconate by the hydrolysis of phosphogluconolactonase. In the third reaction, catalyzed by 6-phosphogluconate dehydrogenase (6PGDH), the 6-phosphogluconate decarboxylates and generates a second NADPH and ribulose-5-phosphate (Ru5P), which in turn converts to ribose-5-phosphate (R5P). The nonoxidative branch is composed of a series of reversible reactions that recruit other glycolytic intermediates like fructose-6-phosphate (F6P) and glyceraldehyde-3-phosphate (G3P) into pentose phosphates.

Enzymes in the PPP are allosterically regulated by their own end products and other metabolites. The allosteric regulation of enzymes and the reversible nature of the nonoxidative PPP branch enable the PPP pathway is well adapted for the metabolic demands of cells in different modes.

For example, in cells for which sustaining redox homeostasis is of higher priority than nucleic acid synthesis, the PPP is adjusted to enhance the oxidative branch and to guide the nonoxidative branch towards re-synthesizing F6P, which in turn converts back to G6P and replenish the oxidative branch. In rapidly dividing cells, nucleic acid synthesis is of higher priority. Most of the pentose phosphates building blocks needed for DNA synthesis are derived from the PPP. Therefore, the PPP will accelerate oxidative branch to generate pentose phosphates from both G6P and enhance the nonoxidative branch to generate F6P and G3P.

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 of metabolites in pentose phosphate pathway metabolites.

Platform

• HPLC-MS/MS

Summary

• Identification and quantification of metabolites in Pentose Phosphate Pathway by HPLC-MS/MS

Sample Requirement

• Normal Volume: 100ul plasma; 50mg tissue; 2e7 cells
• Minimal Volume: 50uL plasma; 30mg tissue; 5e6 cells

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.

With integrated set of separation, characterization, identification and quantification systems featured with excellent robustness & reproducibility, high and ultra-sensitivity, Creative Proteomics provides reliable, rapid and cost-effective pentose phosphate pathway metabolites targeted metabolomics services.