Measurement of the heparin neutralizing capacity of protamine

Several methods of estimating the heparin neutralizing capacity of protamine were investigated for their reliability and practicability. The results from two chemical methods were compared with those from two in vitro biological assays, one of which was the method of the British Pharmacopoeia (1973). An in vivo method using mice was used to assess the accuracy of the in vitro test methods. Three standard heparin preparations were tested against the W.H.O. 1st International Reference Preparation for Protamine. Two of the heparin preparations were of mucosal origin, one of which was the 3rd International Standard, and the third heparin preparation was of lung origin (the 2nd International Standard for Heparin). The mean neutralization values (all methods) of heparin by protamine for a House Reference Preparation and the 3rd and 2nd International Standards for Heparin were 95.8, 109.8 and 89.9 units per mg of Protamine, respectively. All methods read the House Reference Preparation to have a lower value than the 3rd International Standard, which had a higher value than the 2nd International Standard for Heparin. There was a constant relationship between the results of any one method and those of another. The chemical and in vitro biological methods gave results of comparable precision although the latter required a greater degree of technical skill and time to perform.