Protein Binding & Blood Partitioning

Protein Binding and Blood To Plasma Ratio Cell Partitioning Assay

Understand how test molecule interacts with whole blood and plasma proteins.

Knowing a drug’s potential for partitioning is critical in situations where the test drug has a high LogP (lipophilic). In this situation, a significant portion of the drug may be taken up and held by red blood cells (RBCs). For this reason, more informative data describing drug distribution in blood can be obtained by determining both the blood partitioning ratios and direct protein binding.

When the combined knowledge of protein binding and blood partitioning is understood, investigators can make a better determination about whether they should collect whole blood, plasma, or serum for assaying pharmacokinetic behavior of the drug of animal or clinical ADME studies.

Protein Binding assay overview

Equilibrium dialysis or an equivalent is used to separate bound drug (drug-protein) from unbound (free) drug. Whenever possible, Teflon coated compartments are used to reduce nonspecific adsorption. The microdialyzer is agitated during incubation to optimize diffusion kinetics. Plasma pH is adjusted to 7.4 and drug is added and mixed. After the binding period, an aliquot of the plasma-drug mix is placed in the dialysis system and dialyzed against phosphate buffered saline at 37oC with agitation for 2-3 hr. Following the incubation, periodsamples are transferred to Teflon cups for analysis by LC/MS/MS (Lin et al., 2005).

Distribution of drug in whole blood overview

To determine the potential for a drug or chemical to partition into red blood cells (RBC partitioning), a series of drug concentrations (e.g. 20, 10, 5, 2.5 µg/mL) are prepared by making dilutions into 6 mL of whole blood. The packed cell volume (PVC) of the test blood is determined. Once the blood-drug dilutions are made, the samples are incubated for 24 hours at 37°C. After incubation, plasma and RBCs are separated by centrifugation of whole blood samples at 1200 rpm for 10 minutes. Aliquots of whole blood (after incubation), plasma, RBC pack and standard dilutions are analyzed by LC/MS/MS to determine the amount of drug present in each fraction. The concentration of drug is estimated based on calibration curves. Final results represent the average from triplicate experiments (Deshmukh et al., 2009).

Distribution of drug in whole blood overview

Determining packed cell volume or the percentage of RBCs in the blood sample (PCV) is calculated as follows:

A sample of the test blood is taken up into a capillary (microhematocrit) tube and then centrifuged at 10,000 rpms for 5 min at room temperature. This separates the blood into layers.

The volume of packed red blood cells divided by the total volume of the blood sample gives the PCV. The amount of test material in the RBC fraction and Plasma fraction is determined using a calibration curve combined with LC/MS/MS. More accurate partitioning is achieved by using the percent of RBCs in blood (PCV), therefore PCV is determined and used for final calculations.

Percentage of RBC penetration is calculated based upon this value.

Calculated concentration in RBC=Observed concentration in RBC×PCV%
Calculated concentration in Plasma=Calculated concentration in Plasma×Plasma%
Further, extent of partitioning of drug will be determined by using following formulas:

K e/p =Concentration of drug in RBC
Concentration of drug in plasma

K b/p =Concentration of drug in Whole Blood
Concentration of drug in plasma

3 K e/p = Erythrocyte to plasma ratio
K b/p = Whole blood to plasma ratio

Hinderling PH. (1997) RBC- A neglected but important compartment in
pharmacokinetics. Pharm Rev. 49:279–95.

Lin ZJ, Musiano D, Abbot A, Shum L. (2005) In vitro plasma protein binding
determination of flunarizine using equilibrium dialysis and liquid chromatographytandem
mass spectrometry. J Pharm Biomed Anal. 37(4):757-62.

P.V. Deshmukh, P.C. Badgujar, M.M. Gatne (2009) In-vitro red blood cell partitioning of
doxycycline. Indian J Pharmacol. 2009 41(4): 173–175.