Measurements of the kinetics of the pentose phosphate pathway (PPP) in perfused hearts and livers, using [U-13C6]gluconolactone
Bian, F; Minkler, PE; Hoppel, CL; Stanley, WC; Brunengraber, H
HERO ID
4943536
Reference Type
Meetings & Symposia
Subtype
Abstract
Year
2007
Language
English
| HERO ID | 4943536 |
|---|---|
| Year | 2007 |
| Title | Measurements of the kinetics of the pentose phosphate pathway (PPP) in perfused hearts and livers, using [U-13C6]gluconolactone |
| Authors | Bian, F; Minkler, PE; Hoppel, CL; Stanley, WC; Brunengraber, H |
| Abstract | Heinen et al reported the use of [U-13C6]gluconolactone to trace the PPP in microorganisms (Appl. Environ. Microbiology 07/2006, P 4743). We adapted this technique for use in perfused hearts and livers from rats. The tissue was extracted in acetonitrile:water (50:50) and the supernatant applied to an ion exchange solid phase extraction cartridge. The sugar phosphates were eluted with 50 mM ammonium formate in 50% methanol-water (recovery was 90%). We used a hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems) coupled with HPLC to monitor the sugar phosphates using the enhanced resolution mode. We used hydrophilic interaction liquid chromatography to avoid ion pairing reagents and to increase the detection sensitivity. The flux through the PPP is calculated as (uptake of [U-13C6]gluconolactone)/(M6 enrichment of 6-P-gluconate). The latter can be used in tracer amounts because of low background of analytes at M6 and M5. The production of NADPH in the PPP is equal to twice its flux. The contribution of the oxidative branch of the PPP is the enrichment ratio (M5 ribose-5-P)/(M6 6-P-gluconate). Supported by NIH Roadmap grant 5 R33 DK070291. |
| Material Type | Abstract |
| Dupe Override | No |
| Conference Location | Washington, DC |
| Conference Name | Experimental Biology 2007 Annual Meeting |
| Conference Date | APR 28-MAY 02, 2007 |
| Is Public | Yes |
| Language Text | English |