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0.70

0.60

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0.20

u. 0.10 fo-u.

0

18

16

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I

25

o

00 0

o o

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0853 0 o o

§O 'tJ 0 o fP 0

o o o 0

o J ...L I J

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AAG conc. (mg/l00ml)

o

o 0 o

o 0

o @ 0

o ~O cg 0

~ 0 0

~ CO 0 0 1 0 I I I

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o

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AAG conc. (mg/l00ml)

Fig. 1. Oisopyramide free fraction (FF), molar binding ratio (OP/ 0) and ",·acid glycoprotein (AAG) concentrations determined in patient serum specimens (r = -0.79 and 0.86, respectively).

trations were measured in duplicate by enzyme im­munoassay (Syva Co., Palo Alto). The protein binding procedure had a coefficient of variation less than 10%. Disopyramide free fraction averaged 0.25, 0.24, 0.27 and 0.29 in patient serum speci­mens obtained 2, 4, 8 and 12 weeks following transplant surgery, and averaged 0.28 in normal serum. ai-Acid glycoprotein (AAG) concentrations averaged 111, 103,95 and 94mg/100ml in patient serum specimens obtained 2, 4, 8 and 12 weeks

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following transplant surgery, and averaged 45mg/ 1 DOml in normal serum.

Disopyramide free fraction and molar binding ratio (ratio of bound to free drug) were related to serum AAG concentration (fig. 1). The present study demonstrated that: (1) the observed varia­bility in disopyramide binding in patient serum was related to variable serum concentrations of AAG; (2) disopyramide binds extensively to AAG in patient serum; and (3) attempts to define a 'thera­peutic' concentration range with respect to meas­ured total (free plus bound) disopyramide serum concentrations may have little meaning for patients with rapidly changing or elevated serum AAG con­centrations.

Effect of Several Variables on the In Vitro Binding of Disopyramide to Serum Proteins

Leslie M. Shaw, Roy Altman, Bernard C. Thompson and Leona Fields Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia. Pennsylvania, SKCL, King of Prussia, Pennsylvania, and Medical College of Georgia, Augusta, Georgia, USA

We investigated the effects of pH, temperature, assay methods (EMIT® and HPLC), disopyramide concentration, and the separation technique (ultra­filtration vs equilibrium dialysis) on the binding of disopyramide to serum proteins. Using equilib­rium dialysis at 3rC and a disopyramide concen­tration of 3 f.Lg/ml, the average free 14C-disopyr­amide concentration decreased from 1.32 f.Lg/ml to 0.95 f.Lg/ml as pH was increased from 7.0 to 7.8. Although for many drugs the interaction with binding proteins is an exothermic one - with bind­ing decreasing as temperature is increased - changes in temperature had little effect on the free 14C-di­sopyramide concentration. The slopes of the 3 lines derived from plots of free disopyramide concen­tration vs temperature (4°, tOo, 15°, 24°, 3r, and

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4YC) at 2.0, 5.0 and 7.5 !J.g/ml and pH 7.4 were: 0.0004, 0.0031 and 0.0058, respectively, using equilibrium dialysis. Good agreement was ob­tained between EMIT and HPLC measurements of disopyramide in ultrafiltrates prepared from 45 sera by ultrafiltration (Shaw et al., 1982):

Y(HPLC) = 0.92 (EMIT) + 0.00 [SEE = 0.09; r = 0.97].

We confirmed previous observations of a strong dependence of percentage free disopyramide on total drug concentration. Using ultrafiltration, the percentage free disopyramide increased from 22.2 to 54.2% as the total concentration of disopyram­ide, added to aliquots of a serum pool, was in­creased from 2 to 5 !J.g/ml. The variability of the binding of disopyramide to serum proteins was further established for 50 cardiac patients' sera with total disopyramide values ranging from 0.5 to 5.8 /lg/ml. Serum pH was adjusted to 7.4 just prior to preparation of the free drug fraction. The percent­age free disopyramide ranged from 16.5 to 54%.

Relationships between Steady-State Warfarin Concentrations and Anticoagulant Effect

Dennis Mungall. Thomas M. Ludden. James Marshall, Michael Crawford and David Hawkins College of Pharmacy, The University of Texas at Austin; and Departments of Pharmacology and Medicine, The University of Texas Health Science Center, San Antonio, Texas, USA

Limited information is available concerning the relationship between steady-state free and total warfarin concentrations and warfarin's anticoagu­lant effect. Characterisation of such relationships could yield new guidelines for the design of war­farin dosage regimens.

The total warfarin concentration was measured in 385 plasma samples, 1 sample per clinic visit,

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Comparison of free disopyramide measured in 50 patients' sera by both ultrafiltration (UF) and equi­librium dialysis (ED) and the EMIT assay gave the regression line Y(UF) = 1.67 (ED) - 0.067 [SEE = 0.06; r = 0.96].

On average, the equilibrium dialysis values were 39% lower than ultrafiltration values. This is most likely due to a major difference in these tech­niques, namely, the volume occupied by free drug in the equilibrium dialysis method is twice that oc­cupied in the original sample and in the ultrafil­tration method. Since the free concentrations ob­tained by ultrafiltration of the equilibrium dialysis retentate were comparable with those measured in the dialysate by the EMIT method for 30 patients' sera [Y (UF, retentate) = 1.05 X (ED, dialysate) + 0.042], the ultrafiltration method does not signifi­cantly perturb the actual free disopyramide con­centration.

Reference Shaw. L.M. et al.: Clinical Chemistry 28: 1589 (1982).

from 133 outpatients. Total concentrations (CT )

were assayed using high performance liquid chro­matography (Fasco et al., 1977). Prothrombin times were attained at each visit and converted to pro­thrombin complex activity, utilising the equation of Sheiner (1969). The free (unbound) warfarin concentration (Cd was also measured in 74 sam­ples from 50 of these patients. Ultrafiltration was used to determine the free fraction (EMIT® FreeLevel™ System 1, Syva Co., Palo Alto) util­ising a chromatographic method for eliminating radiochemical impurities (Mungall et al., 1983).

Results: No significant correlation was found between the logarithm of the dose (mgfday/m2) and percentage suppression of prothrombin complex activity (r = -0.099, p > 0.05). Significant corre­lations between percentage suppression of pro­thrombin complex activity and the logarithm of both total (r = 0.45, p < 0.05) and free (r = 0.5, p < 0.05) warfarin concentrations were found. Table I summarises the relationship between total