Plasma protein binding of drugs

In blood many drugs are partly associated with blood constituents such as plasma proteins and erythrocytes. For most such drugs, only the unbound fraction of the total amount is available for diffusion out of the vascular system to sites of pharmacological activity. The unbound fraction of drug in plasma is therefore regarded as pharmacologically active, while the fraction bound to plasma protein is considered pharmacologically inactive. We studied some aspects of plasma protein binding of drugs, in particular regarding their implications on therapeutic drug monitoring. Evacuated blood collection tubes were reported in the literature to disturb drug monitoring by interference with plasma protein binding of certain drugs, resulting in an increased drug uptake by red blood cells and a decreased total plasma or serum concentration. We investigated the influence of various brands of evacuated blood collection systems on therapeutic drug monitoring (the old type, red stoppered Vacutainer| the new type, blue stoppered Vacutainer| Monoject | and Venoject| No disturbance was found in the assay of ethosuximide, phenobarbital, phenytoin, valproic acid, digitoxin, digoxin, procainamide, gentamicin and theophylline. Using Monoject | and old type Vacutainer | tubes, lower levels were found in the disopyramide assay: 91.3-+4.6% (p < 0.05) and 91.7_+7.o% (not significant), respectively, and in the quinidine assay: 82.8+6.7% (p < o.o2) and 83.9+4.4 % (p < o.ooI), respectively, as compared with glass tubes. In the carbamazepine assay a decrease was found in the Monoject | tubes only: 93.7-+I.7% (P < o.o0. The stoppers of the Monoject | tubes and the old type Vacutainer | tubes contained the plasticizer tris(2-butoxyethyl)-phosphate (TBEP), which has been shown to be a potent inhibitor of the binding of several drugs to cq-acid glycoprotein. Using the new type Vacutainer* and the Venoject | no interferences were found. In routine therapeutic drug monitoring, a measurement of the total (bound plus unbound) concentration of drug in serum or plasma is used to evaluate the safety and effectiveness of the drug dosage regimen. Problems for the interpretation of these levels can arise when plasma binding is disturbed. Measurement of free drug plasma concentrations represents therefore a refinement in therapeutic drug monitoring, and may become the preferred means of monitoring therapy with certain drugs. Some pitfalls in the methods used for measuring plasma protein binding of drugs were studied. A variety of techniques have been developed to measure unbound drug concentrations, among which equilibrium dialysis and ultrafiltration are most commonly applied. We tested the usefulness of three simple, commercially available ultrafiltration systems (Centriflo | CF25, Centriflo | CF5oA and Ultrafree | in determining concentrations of unbound carbamazepine in plasma. The concentrations of carbamazepine measured in the first fractions of the ultrafiltrate were low, probably as a result of binding of the drug to the ultrafiltration system. The amount of this binding decreased in the order Centriflo CF25 > Centriflo CF5oA > Ultrafree. When using the Centriflo cones, the first portion of ultrafiltrate obtained after centrifuging for 15 min, must therefore be discarded. Using the Ultrafree filters this was not necessary. Plasma proteins were found to pass through the Ultrafree filter membrane when more than 7oo gl ultrafiltrate was collected. Comparison of the ultrafiltration methods with equilibrium dialysis showed a good correlation: r = 0.96 (CF25), r = 0.97 (CF5oA), and r = o.98 (Ultrafree). If equilibrium dialysis represents true free levels, Ultrafree filters gave values with good accuracy and precision. The Centriflo filters, however, produced systematically and significantly lower free carbamazepine concentrations, even if the first fraction of ultrafiltrate was discarded in order to decrease binding effects. The free fractions measured were: o.28-o.3o (equilibrium dialysis), o. I7-O. I8 (CF25) , 0.22-0.24 (CF5oA) and 0.29-0.30 (Ultrafree). The time required to collect the ultrafiltrate was 3o min (Centriflo cones) and 2 h (Ultrafree). If equilibrium dialysis is used to estimate plasma