Thromboxane radioimmunoassay

Granstrom E Sources of error in prostaglandin and Thromboxane radioimmunoassay. In Radioimmunoassay of Drugs and Hormones Albertini A, Da Prada Peskar BA (eds)/ Amsterdam-New York-Oxford, Elsevier/North Holland Biomedical Press, 1979, p 229-238. 

An alternative solution is to abandon bioassay entirely and use a more specific approach instead. One such approach in TXA 2 assay is based on the rapid conversion of this unstable compound into a stable derivative by trapping with excess methanol or some other nucleophilic reagent (see above. Elucidation of the thromboxane structure). If methanol is used, the formed products are two epimers of mono-O-methyl TXB2 (Fig. 2). These compounds are biologically inactive but are instead suitable for assay by methods requiring chemical stability of the measured compound, such as gas chromatography mass spectrometry or radioimmunoassay. 

By far the most common method in this field is radioimmunoassay. After the first radioimmunoassay for TXB2 was published [227], a very large number of similar assays were developed [e.g. 217,228-232]. This type of measurement has been used for detection and/or quantification of thromboxane synthesis in platelet preparations, tissue homogenates, organ perfusates, cell culture media, various exudates or other biological material it has also been used in monitoring enzyme activity of thromboxane synthetase during purification procedures. 

Each type of assay method (e.g. bioassay, gas chromatography mass spectrometry, radioimmunoassay) suffers from certain sources of error, which may seriously influence the final results. Some bioassay problems, specific for TXAj measurements, were briefly discussed above. In addition to such sources of error, however, there are also other pitfalls in the three different approaches to thromboxane measurements described above (i.e. direct and indirect assay of TXA2, and assay of TXB2, respectively). 

The most striking example of this is plasma fatty acids, which endogenously occur in plasma in concentrations around 200 yg/ml. In comparison, the levels of several prostaglandin metabolites are around 50 pg/ml or less. Although crossreaction with straight chain fatty acids is always very low in prostaglandin or thromboxane assays, the high amounts present in the plasma would definitely interfere in the radioimmunoassay, if the fatty acids occurred free in the sample (28). Thus, unless the samples are not extensively purified after extraction to remove the fatty acids, a simple extraction procedure cannot be recommended. The possible influence of fatty acids in radioimmunoassay has recently been pointed out (29). 

The identities of the majority of these disturbing factors are not known. They are probably not chemically related to prostaglandins or thromboxanes, and their interference in radioimmunoassays is not simply caused by cross-reaction. Instead the antigen-antibody binding is probably non-immuno-logically inhibited by many of these substances. One compound which has caused a lot of problems in analytical work is the ubiquitous impurity bis(2-ethyl-hexyl)phthalate ( di-... 

Granstrom E, Kindahl H, Samuelsson B A method for measuring the unstable thromboxane A2 Radioimmunoassay of the derived mono-0-methyl-thromboxane B2. Prostaglandins 12 929-941, 1976. 

Results are similar when one aggregates washed platelet suspensions with PGH2. Exogenous addition of the immediate substrate for the thromboxane synthetase bypasses the cyclooxygenase enzyme, but TxB2, measured by radioimmunoassay accumulates, and aggregation accompanies the accumulation [Figure 2]. 

Both PASs and Biomer solutions (10%) were poured individually into glass tubes, and the insides of the glass tubes (7-mm ID X 50 mm) were coated, the solvent was then evaporated under vacuum at 60°C for 48 h. One milliliter of human whole blood was poured into the tubes and allowed to stand for 7 min. After centrifuging (4000 rpm, 30 min), 300 jxL samples of separated plasma were collected and thromboxane B2 (TXB ) concentrations were measured by radioimmunoassay (RIA) [44,45]. 

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