Summary
The analytical basis of membrane separation in determining plasma free drug concentrations is developed from consideration of the generalised ideal mass action and conservation laws applied to thermodynamic states of differing component volumes. The intuitively surprising theoretical independence of free drug concentration with changing fractional volume of protein predicts the equivalence of final free drug concentration after uniform pre-dilution with that after equilibrium dialysis against the same volume of buffer. Similarly, the ultrafiltrate free concentration of drugs in ideal plasma binding equilibrium is theoretically constant, regardless of the extent of reduction in fractional volume of binding protein during filtration.
The pitfalls in calculating free and bound fraction and concentration from equilibrium dialysis are reviewed and expressions presented to correct the major artefacts. The dilution behaviour of drugs is predicted from the normalised mass action model. Practical aspects of validating ultrafiltration free drug levels using equilibrium dialysis are outlined.
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References
Behm, H.L. and Wagner, J.G.: Errors in interpretation of data from equilibrium dialysis protein binding experiments. Research Communications in Chemical Pathology and Pharmacology 26: 145–160 (1979).
Boyer, P.D.; Ballou, G.A. and Luck, M.J.: The combination of fatty acids and related compounds with serum albumin III. The nature and extent of the combination. Journal of Biological Chemistry 167: 407–424 (1947).
Builder, S.E. and Segel, I.H.: Equilibrium ligand binding assays using labeled substrates: Nature of the errors introduced by radiochemical impurities. Analytical Biochemistry 85: 413–424 (1978).
Bush, M.T. and Alvin, J.D.: Characterization of drug-protein interactions by classic methods. Annals of the New York Academy of Sciences 226: 36–43 (1973).
Chadwick, C; Phipps, D.A. and Powell, C: Potential methodological errors in the application of equilibrium dialysis to the determination of “free” and “bound” plasma tryptophan. Clinica Chimica Acta 99: 279–282 (1979).
Chignell, C.F.: Application of physicochemical and analytical techniques to the study of drug interactions with biological systems. Chemical Rubber Corporation Critical Reviews in Toxicology 1: 413–465 (1972).
Christensen, J.H.; Andreasen, F. and Jensen, E.B.: The binding of thiopental to serum proteins determined by Ultrafiltration and equilibrium dialysis. Acta Pharmacologica et Toxicolol-ogica 47: 24–32 (1980).
Craig, L.C.: Differential dialysis. Advances in Analytical Chemistry and Instrumentation 4: 35–74 (1965).
Gilbert, G.A. and Jenkins, R.C.LI.: Sedimentation and electro-phoresis of interacting substances. II. Asymptotic boundary shape for two substances interacting reversibly. Proceedings of the Royal Society A. 253: 420–437 (1959).
Greenberg, D.M.: The interactions between the alkali earth cations, particularly calcium, and proteins. Advances in Protein Chemistry 1: 121–151 (1944).
Greenberg, D.M. and Greenberg, M.; Ultrafiltration, I. Ultrafiltration of electrolytes from alkali caseinate solutions. Journal of Biological Chemistry 94: 373–382 (1931–1932).
Greenberg, D.M. and Gunther, L.: On the determination of diffusible and non-diffusible serum calcium. Journal of Biological Chemistry 85: 491–508 (1929–1930).
Hinderling, PH.; Bres, J. and Garrett, E.R.: Protein binding and erythrocyte partitioning of disopyramide and its monodeal-kylaed metabolite. Journal of Pharmaceutical Sciences 63: 1684–1690 (1974).
Jung, D.; Mayersohn, M. and Perrier, D.: The “ultra-free” ultra-filtration technique compared with equilibrium dialysis for determination of unbound thiopental concentrations in serum. Clinical Chemistry 27: 166–168 (1981).
Keen, P.M.: The binding of penicillins to bovine serum albumin. Biochemical Pharmacology 15: 447–463 (1966).
Kinget, R.; Bontinck, A.M. and Herbots, H.: Problems of dialysis techniques in the study of macromolecule binding of drugs. International Journal of Pharmaceutics 3: 65–72 (1979).
Klotz, I.M. and Hunston, D.L.: Protein interactions with small molecules. Journal of Biological Chemistry 250: 3001–3009 (1975).
Kurz, H.; Trunk, H. and Weitz, B.: Evaluation of methods to determine protein-binding of drugs. Equilibrium dialysis, Ultrafiltration, ultracemrifugation, and gel filtration. Drug Research 27: 1373–1380 (1977).
LeComte, M.; Zini, R.; d’Athis, P. and Tillement, J.P.: Phenytoin binding to human albumin. European Journal of Drug Metabolism and Pharmacokinetics 1: 23–28 (1979).
Lunde, P.K.M.; Rane, A.; Yaffe, S.J.; Lund, L. and Sjöqvist, F.: Plasma protein binding of diphenylhydantoin in man. Interaction with other drugs and the effect of temperature and plasma dilution. Clinical Pharmacology and Therapeutics 11: 846–855 (1970).
MacMahon, W.; Thompson, J.; Bowers, W. and Sgoutas, D.: A simplified ultrafiltration method for determination of serum free Cortisol. Clinical Chimica Acta 131: 171–184 (1984).
McNamara, P.J. and Bogardus, J.B.: Effect of initial conditions and drug-protein binding on the time to equilibrium in dialysis systems. Journal of Pharmaceutical Sciences 71: 1066–1068 (1982).
Monot, C; Netter, P.; Stalars, M.C.; Martin, J.; Royer, R.J. and Gaucher, A.: Difficulties in applying the Scatchard model of ligand binding to proteins — proposal of new mathematical tools — Application to salicylates. Journal of Pharmaceutical Sciences 72: 35–41 (1983).
Munson, P.J. and Rodbard, D.: Ligand: A versatile computerized approach for characterization of ligand-binding systems. Analytical Biochemistry 107: 220–239 (1980).
Nimmo, I.A.; Atkins, G.L.; Strange, R.L. and Percy-Robb, J.W.: An evaluation of ways of using equilibrium and dialysis to quantify the binding of ligand to macromolecule. Biochemical Journal 165: 107–110 (1977).
Oppenheimer, J.A. and Surks, M.I.: Determination of free thryroxin in human serum: A theoretical and experimental analysis. Journal of Clinical Endocrinology 24: 785–793 (1964).
Paulus, H.: A rapid and sensitive method for measuring the binding of radioactive ligands to proteins. Analytical Biochemistry 32: 91–100 (1969).
Paxton, J.W. and Donald, R.A.: Concentrations and kinetics of carbamazepine in whole saliva, parotid saliva, serum ultra-filtrate, and serum. Clinical Pharmacology and Therapeutics 28: 695–702 (1980).
Porter, R.J. and Layzer, R.B.: Plasma albumin concentration and diphenylhydantoin binding in man. Archives of Neurology 32: 298–303 (1975).
Robbins, J. and Johnson, M.L.: Theoretical considerations in the transport of the thyroid hormones in blood; in Ekins et al. (Eds) International Symposium on Free Level Thyroid Hormones, pp. 1–16 (Elsevier North Holland, New York 1979).
Rosner, W.: Some theoretical considerations regarding filter disk assays. Analytical Biochemistry 67: 422–427 (1975).
Rowland, M. and Tucker, G.: Symbols in pharmacokinetics. Journal of Pharmacokinetics and Biopharmaceutics 8(5): 497–507 (1980).
Selikson, M.; Gibson, R.E.; Eckelman, W.C. and Reba, R.C.: Calculation of binding isotherms when ligand and receptor are in different volumes of distribution. Analytical Biochemistry 108: 64–71 (1980).
Sjöholm, I.; Kober, A.; Odar-Cederlöf, I. and Borgå, O.: Protein binding of drugs in uremic and normal serum: The role of endogenous binding inhibitors. Biochemical Pharmacology 25: 1205–1213 (1976).
Smith, J.B. and Jubiz, W.: A source of error in equilibrium dialysis. Steroids 36: 393–403 (1980).
Sophianopoulos, J.A.; Durham, S.J.; Sophianopoulos, A.J.; Ragsdale, H.L. and Cropper, W.P.: Ultrafiltration is theoretically equivalent to equilibrium dialysis but much simpler to carry oui. Archives of Biochemistry and Biophysics 187: 132–137 (1978).
Steinhardt, J. and Reynolds, J.A.: Multiple equilibria in proteins, pp. 34–53. 79–83 (Academic Press, New York 1969).
Suter, P. and Rosenbusch, J.P.: Determination of the Donnan effect in equilibrium dialysis and a simple method for its correction. Analytical Biochemistry 82: 109–114 (1977).
Van der Giesen, W.F. and Wilting, D.: New aspects of free drug concentration determination in human serum with equilibrium dialysis. Consequences of the binding properties of albumin for the determination of free warfarin concentrations in serum. Methods and Findings in Experimental and Clinical Pharmacology 4: 417–423 (1982).
Vlahos, I.; MacMahon, W.; Sgoutas, D.; Bowers, W.; Thompson, J. and Trawick, W.: An improved ultrafiltration method for determining free testosterone in serum. Clinical Chemistry 28: 2286–2291 (1982).
Whitlam, J.B. and Brown, K.F.: Ultrafiltration in serum protein binding determinations. Journal of Pharmaceutical Sciences 70: 146–150 (1981).
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Bowers, W.F., Fulton, S. & Thompson, J. Ultrafiltration vs Equilibrium Dialysis for Determination of Free Fraction. Clin Pharmacokinet 9 (Suppl 1), 49–60 (1984). https://doi.org/10.2165/00003088-198400091-00007
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DOI: https://doi.org/10.2165/00003088-198400091-00007