Gräbner R
Exp Pathol (Jena). 1977;13(1):68-77.
The anorectic drug chlorphentermine (Chlph) has been reported to cause lipoidosis-like cellular alterations in many organs, especially in lungs. A weak inflammation has been observed during the first week of daily application. After that time a pronounced foam cell production with many lamellated inclusion bodies in the cell occurs. It has been supposed that this action is caused by an inhibition of phospholipid-degradation due to an association between the drug and phospholipids. To substantiate this mechanism an in vitro study a bout the action of Chlph on the hydrolysis of phosphytidyl choline (PC) by phospholipase A2 (bee venom) was undertaken.
Pur PC was obtained by column chromatography of egg lecithin. PC was used in two physical states. Handshaken liposomes are used as a model of the lamellated inclusion bodies of drug- induced phospholipidosis. The kinetic analysis was carried out on single bilayered liposomes obtained by injection of an ethanolic PC-solution into 0.16 M KCl. Purified bee venom was used as enzyme source. Usually the drug was added before the initiation of the enzyme reaction. In some cases Chlph was added after starting the hydrolysis by phospholipase for a detailed characterization of the type of interaction between Chlph and PC. The velocity of the enzyme reaction was measured by pH- stat titration and was expressed as mM H+-release per min.
Two phases of Chlph-action are observed. A time limited stimulation of hydrolysis occurs immediatly after addition of the drug. The enzyme reaction is inhibited after the disappearence of this activation. This inhibition is independent of the physical state of the substrate and is very pronounced at equimolar mixtures of Chlph and PC (88 per cent inhibition in handshaken liposomes; 78 per cent inhibition in single bilayered liposomes). At inhibitor concentrations below 10 mol per cent the hydrolysis is not affected. By kinetic analysis it was found that the inhibitory action is due to an association between the inhibitor and the substrate. The Lineweaver-Burk- and Dixon-replots show a series of curves characteristic for this type of interaction (concave shape; no common intersections, situated in the 2nd quadrant). The intermediate stimulation of the substrate hydrolysis occurs only during the reaction of Chlph with PC. This is concluded from the following observations: The duration of activation is proportional to the inhibitor concentration as well as to the substrate concentration, i.e. it is proportional to the concentration of both reactants. The activation does not occur if the enzyme reaction is started some time after mixing inhibitor and substrate, i.e. after finishing the reaction. The results are discussed in relation to the in vivo action of Chlph.
