A Simple Method (TBA-method) for Estimation of Arachidonate Metabolism by Human Platelets
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The arachidonic acid (AA) metabolism of human platelets was studied under various experimental conditions by use of the thiobarbituric acid (TBA) reaction as well as a radioisotope technique, and a simple method was developed for estimation of exogenous and endogenous AA metabolism including platelet cyclo-oxygenase (PCO) pathway, platelet lipoxygenase (PLO) pathway and AA liberation from membrane phospholipids.<BR>When AA was incubated with washed platelets cyclo-oxygenase of which had been completely inhibited by aspirin (ASA), TBA-reactive substance (TBARS) was not detected within short incubation periods, especially in alkaline media, although control (intact) platelets produced promptly a significant amount of TBARS even under such experimental conditions. Therefore, it was suggested that TBARS produced by the incubation of AA with control platelets for short incubation periods before the development of ASA-resistant TBARS in alkaline media was malondialdehyde (MDA) via the PCO pathway. When AA was incubated with the cyclo-oxygenaseinhibited platelets, prolongation of the incubation time especially in acid media produced a significant amount of TBARS, suggesting that TBARS thus obtained was produced via the PLO pathway.<BR>These concepts were supported by the following experimental results: (1) When <SUP>14</SUP>C-AA was incubated with platelets, time courses for the formation of PCO products were faster than those for the formation of PLO products and optimal pH values for the formation of PLO products were around 6 while those for PCO products were around 7.4;(2) Time course and pH profile for the production of TBARS by the incubation of AA with the ASA-treated platelets were similar to those obtained by the incubation of AA with the soluble fraction of platelet homogenates (PLO enzyme preparation);(3) Phenidone, a PLO enzyme inhibitor, inhibited the production of TBARS in both of these experiments, indicating that TBARS thus obtained was synthesized from AA by the PLO enzyme. When thrombin was incubated with washed platelets, TBARS was promtly produced and optimal pH values for its production were around 8. The TBARS formation by thrombin was completely inhibited by ASA, indicating that the thrombininduced TBARS (Th-TBARS) was MDA via the PCO pathway alone derived from endogenous AA liberatied by thrombin from platelet phospholipids. Therefore, Th-TBARS can be used as an indicator for estimating AA liberated from phospholipids when the PCO pathway is normal.<BR>Based on these experimental results, the PLO and PCO pathways were estimated by TBARS determined after the incubation of 0.2mM AA at pH 6.5 for 10 min with 10<SUP>8</SUP> platelets which had been incubated with 1mM ASA for 5 min at 37° and after the incubation of 0.2mM AA with 10<SUP>8</SUP> untreated platelets at pH 7.4 for 1 min, respectively. AA liberation in cases of normal PCO pathway was estimated by TBARS determined after the incubation of thrombin (10U/ml) with 2×10<SUP>8</SUP> platelets at pH 7.4 for 10 min. Normal values expressed in terms of nmol MDA were 1.17±0.34 per 108 platelets (m±SD, n=31), 0.79±0.15 per 108 platelets (n=31) and 0.64±0.24 per 2×108 platelets (n=23) for the PLO pathway, PCO pathway and AA liberation, respectively. The validity of the experimental conditions for estimation of the PLO and PCO pathways was confirmed by the results that PLO and PCO activities as determined by those conditions were not detected when platelets were obtained from PLO defective patients and from ASA-ingested normal subjects, respectively, and that TBARS production by normal platelets as an indicator of PLO and PCO pathways was strongly inhibited in vitro by phenidone and ASA, respectively.
- Japan Society of Clinical Chemistryの論文
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- A Simple Method (TBA-method) for Estimation of Arachidonate Metabolism by Human Platelets