Behaviour of Heparin in vivo - Its Metabolism as Followed by a New 125-I Shpp-Conjugate

Using the reverse sequence of the Bolton and Hunter reagent (SHPP -succinimidylbydroipbenylpropionic acid) than that normally applied for protein labelling we were able to incorporate 0.08 mole SHFP/mole heparin using the non-iodinated molecule. Following purification of the conjugate on protamine-agarose, it could be labelled in a conventional chloramine-T-iodination to yield 125-I or 125-I heparin with a specific activity of 10-20 uCi/ug, more than 95% of the tracer was atill bound to protamine following iodination. Using other chromatography systems, radioactive labelalways coincided with bioassay or chemical assay of heparin. Alkaline hydrolysis studies showed that the label was bound to amino groups of glucosamine and not to a terminal lylosyl-serine residue. In vivo studies shoved that the half life of disappearance was strongly dose dependent, and that the label re-appeared in the circulation after 2 hours and then persisted for a much longer period. This metabolic product was de-sulphated heparin which was biologically inactive but otherwise an intact polysaccharide which appears to be slowly cleared via the kidneys. Whole body scanning of a 131-I-conjugate heparin at +40 mins (when ca 90% of the label had temporarily disappeared from the circulation) shoved that desulphation took place largly in the liver and spleen.