On the relationship between molecular mass and anticoagulant activity in a low molecular weight heparin (enoxaparin).

A low molecular weight heparin (enoxaparin, mean molecular weight approximately 4,400) was separated by gel chromatography into eight different fractions with a narrow distribution around the following mean molecular weights: 1,800, 2,400, 2,900, 4,200, 6,200, 8,600, 9,800 and 11,000. We compared the influence of enoxaparin on the generation of thrombin in plasma to that of the eight fractions. We determined: a) the % of material with high affinity to antithrombin III (HAM) and the % of HAM above the critical chain length necessary to allow for thrombin inhibition (ACLM), b) the specific catalytic activity on the decay of endogenous thrombin, and c) the inhibition of over-all thrombin formation in the extrinsic and the intrinsic pathway. From b and c we calculated the inhibition of prothrombin conversion in these pathways. We found that a) there is a gradual decrease of the HAM fraction with decreasing molecular weight; b) the specific catalytic activity for the inactivation of thrombin does not vary significantly between the fractions when expressed in terms of ACLM; c) the potency to inhibit prothrombin conversion does not vary significantly between the fractions when expressed in terms of HAM.

Determination of 125I-labelled thyroxine glucuronide by reversed-phase high-performance liquid chromatography using on-line radiochemical detection to determine UDPglucuronosyltransferase activity.

A convenient, fast and highly sensitive high-performance liquid chromatographic method, using on-line radiochemical detection, is described for the determination of [125I]thyroxine glucuronide. The method involves direct injection of the supernatant, a total analysis time of 30 min and a detection limit of 1 pmol. The results demonstrate that the method is suitable for the determination of UDPglucuronosyltransferase activity with thyroxine as substrate in native hepatic microsomes. The rate of thyroxine glucuronidation in microsomes from rats treated with Arodor 1254 was ten times higher than in control microsomes, indicating that with this method, increases of UDPglucuronosyltransferase thyroxine activities, often associated with hepatic induction process involved in thyroid hypertrophy, can be easily detected. This method could also be applied to all experimental biological systems that involve the separation and quantification of [125I]thyroxine and [125I]thyroxine glucuronide.