Fluorometric assays of phospholipase A2 activity with three different substrates in biological samples of patients with schizophrenia.
The rationale of this study was to understand the complexity of kinetics of fluorogenic phospholipid substrates as well as contradictory findings of clinical papers measuring phospholipase A2 (PLA2) activity using different methodologies. The aim was to recommend to clinicians and researchers what substrate in conjunction with what assay should be used. Two methods, (i) continuous fluorometric assay and (ii) high performance thin layer chromatography (HPTLC) on microplates combined with quantitative image scanning, were studied with three different substrates (bis-BODIPY FL C11-PC, NBDC6-HPC, PED6). The study demonstrates that NBD-PC is not a suitable substrate to measure PLA2 activity using a spectrofluorometer. On the other hand, NBD-PC gives the highest and most reproducible integrated light intensities (ILls) in HPTLC studies. Slow time-dependent increases in fluorescence intensities recorded with biological samples in fluorometers, but not caused by substrate splitting, had to be classified as "perturbation kinetics". PLA2 activities in blood samples of 26 unmedicated schizophrenia patients and 26 age-matched healthy controls were measured by the spectrofluorometric method and then compared with the activity data obtained with the HPTLC method. A significant group difference was found only with the HPTLC. In order to get more reliable results, we recommend that clinicians and researchers use NBD-phosphatidylcholines as PLA2 substrates in biological samples and start with an analytical separation of reaction products followed by image analysis of the fluorescent spots.
Lasch
Jürgen
J
Department of Physiological Chemistry, Medical Faculty, Martin Luther University of Halle-Wittenberg, Halle/Saale, Germany. juergen.lasch@medizin.uni-halle.de
Willhardt
Ingo
I
Kinder
Daniel
D
Sauer
Heinrich
H
Smesny
Stefan
S
D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D012016:Reference Values; D015203:Reproducibility of Results; D012559:Schizophrenia / Q000201:enzymology*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D010741:Phospholipases A; D054467:Phospholipases A2