Die ontwikkeling van elektrosproei massaspektrometrie vir die bepaling van karnitienpalmitonieltransferase I-aktiwiteit in fibroblaste en leukosiete

Abstract

Camitine Palmitoyltransferase I (CPT I) is one of the important enzymes associated with normal mitochondrial membrane transport of certain metabolites. The impo11ance of the enzyme in normal energy production is well illustrated during fasting conditions when a large flux of long chain fatty acids must be transported over the mitochondrial membrane to undergo fl-oxidation. Up to now CPT I activity has been assayed in various tissues, including liver, leukocytes, platelets and fibroblasts by the use of an isotope exchange forward assay which measures the rate of palmitoyl-L-[ methyl-3H]carnitine formation from palmitoyl-CoA and L-[ methyl- 3H]carnitine. We have developed an electrospray ionization mass spectrometric method for detecting palmitoylcamitine formation from palmitoyl-CoA and camitine, thus avoiding the use of radio labeled isotopes. In this assay, time dependent conversion of free camitine by CPT I to palmitoylcamitine is measured quantitatively, relative to isotopically labelled palmitoylcamitine, by parent ion monitoring of fragment ion m/z 85. The combination of high sensitivity and selectivity of tandem mass spectrometry along with the environment friendly nature of the method makes it an ideal technique to measure CPT I activity. Results of this study were presented at the following local and international congresses: Nolte, L., Van der Westhuizen, F.H., Knoll, D.P., Mienie, L.J., Pretorius, P.J., & Erasmus, E. Camitine palmitoyltransferase I activity monitoring in fibroblasts and leukocytes using electrospray mass spectrometry. South African Academy for Science and Arts Year, Potchefstroom, June 1997. Nolte, L., Van der Westhuizen, F.H., Knoll, D.P., Mienie, L.J., Pretorius, P.J., & Erasmus, E. Carnitine palmitoyltransferase I activity monitoring in fibroblasts and leukocytes using electrospray mass spectrometry. First Symposium of the Biochemistry Society between the Biochemistry Departments of PU for CHE and UOVS, Bloemfontein, June 1997. Nolte, L., Van der Westhuizen, F.H., Knoll, D.P., Mienie, L.J., Pretorius, P.J., & Erasmus, E. Carnitine palmitoyltransferase I activity monitoring in fibroblasts and leukocytes using electrospray mass spectrometry. ih International Congress of Inborn Errors of Metabolism, Vienna, Austria, May 1997. A publiction originating from this work has been accepted: Nolte, L., Van der Westhuizen, F.H., Pretorius, P.J., & Erasmus, E. Carnitine Palmitoyltransferase I Activity Monitoring In Fibroblasts And Leukocytes Using Electrospray Ionization Mass Spectrometry. Analytical Biochemistry. (Accepted and revised, 1997).