Determination of heparin in clinically normal dog gingiva

The purpose of this study was to develop a technique to allow the extraction, identification and quantification of heparin in small samples (0.5 g) of normal gingival tissue of the Beagle dog. Heparin was extracted following homogenization, defattening, proteolytic digestion and precipitation with organic solvents and identified by a microelectrophoretic method carried out on an agarose slide. The concentration of heparin was determined by measuring the optical density of the metachromatic toluidine blue stained spots and comparing them with standard reference heparin spots. Gingiva contained 25 +/- 1.9 units of heparin per gram of wet tissue with a relative migration rate of 0.92 +/- 0.005 vs the unit value for reference heparin. Heparin was further characterized by enzyme degradation with adapted F. heparinum (heparinase). Its anticoagulant activity was demonstrated by extending Plasma Thrombin Time, which was normalized with the anti-heparin substance protamine sulfate.