Thyrotropin releasing factor: radioimmunoassay and distribution in biological fluids and tissues.

The histidine residue of thyrotropin releasing factor (TRF, pGlu-His-Pro-NH2) offers the possibility of conjugating TRF to a macromolecule to produce TRF antibodies. The antibodies thus raised detect changes in pGlu and Pro-NH2 residues and have broadly similar crossreactivity patterns. The histidine residue also provides the possibility of incorporating radioiodine in the molecule. Cation exchange chromatography separates [125I2]-TRF from [125I]-TRF and TRF and produces tracer for TRF radioimmunoassay. Although TRF antisera are specific, many normal tissue constituents and chemical compounds, when present in high concentrations, interfere with TRF radioimmunoassays, leading to artificially high TRF levels. Hence to obtain reliable results the interfering compounds have to be removed before TRF is measured by radioimmunoassay. The quantities of immunoreactive TRF found in various rat tissue extracts are as follows: neonatal pancreas, hypothalamus, posterior pituitary 200-600 pg/mg; brain stem, spinal cord, neonatal hypothalamus 50-200 pg/mg; and cerebellum, cerebrum, placenta, pancrease, intestine 0.1-50 pg/mg. TRF is undetectable (below 0.1 pg/mg) in extracts of peripheral nerves, liver, lungs and kidneys. The bulk of the TRF immunoreactivity in pancreatic and brain extracts elutes in reverse phase high pressure liquid chromatography (HPLC) with gradient elution like synthetic TRF, suggesting that most of the TRF immunoreactivity is due to pGlu-His-Pro-NH2. No firm conclusion can be drawn about the possible function of TRF in extrahypothalamic tissues, and special attention should be given to the characterization of the immunoreactive material.