Separation of Endothelin Isoforms by High Performance Liquid Chromatography and Detection by Radioimmunoassay
All three isoforms of endothelin (ET) have been detected in human and animal plasma and tissues (1, 2, 3, 4). As described in Chapter 2, radioimmunoassay (RIA) and enzyme-linked-immunosorbent assay (ELISA) are the methods of choice to quantify immunoreactive ET peptides. However, ET-1, ET-2 and ET-3 share a common C-terminal sequence and indeed ET-1 and ET-2 differ by only two amino acids in the N-terminal region. Thus, it is usually not possible to distinguish between the different isoforms using available antibodies (see Note 1). In order to determine which isoforms are present in, e.g., tissue, plasma, or cell culture extracts, it is necessary to carry out high performance liquid chromatography (HPLC) separation of the samples. As the levels of ET peptides in biological samples are too low to be detected by ultraviolet (UV) spectrophotometry and would be masked by the presence of other peptides, fractions are collected from the HPLC analytical column for subsequent analysis by RIA as previously described (see Chapter 2). Using this technique, we have identified mature ET-1, ET-2, ET-3 and the precursor, big ET-1, in tissue extracts of human heart (5). We have shown that ET-1 is the major isoform, confirmed by mass spectrometry (6) (see Note 2), present in cultured human endothelial cells from a number of sources, including umbilical vein and the endocardium, whereas levels of ET-2 and ET-3 were below the level of detection in these cells. Absence of ET-2 in endothelial cells is surprising as mRNA encoding this isoform is present in human endothelial cells (7) and the mature peptide is present in plasma (3). The source of ET-2 remains unclear, but it is possible that this peptide is synthesized only under certain, as yet unspecified, physiological or pathophysiological conditions.
KeywordsHigh Performance Liquid Chromatography Acetonitrile Polypropylene Biotin Spectrophotometry
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