Impact of hemolysis during sample collection: how different is drug concentration in hemolyzed plasma from that of normal plasma?

Hemolysis is a common phenomenon in clinical studies. Despite the growing interest in hemolysis matrix effect, how hemolysis impacts the representability of hemolyzed plasma samples was rarely evaluated. The purpose of this research is to perform such an evaluation by theoretical consideration and experiment. A formula for estimating the impact is proposed, which includes the degree of hemolysis and the drug's red blood cell (RBC): plasma concentration ratio. The impact of hemolysis on the representability of hemolyzed plasma samples is compound-dependant. Given the same degree of hemolysis, the stronger a drug binds to RBCs, the more significant the impact of hemolysis. For a drug with high affinity to RBCs, the results of hemolyzed plasma samples may not be useful even though they are accurate. There is an overall agreement between theoretical predication and experimental results. Among the ten different drug compounds tested, only methazolamide, which binds strongly to RBCs, showed significant change in plasma concentration due to hemolysis.

In vitro characterization of the erythrocyte distribution of methazolamide: a model of erythrocyte transport and binding kinetics.

The rate and extent of binding of methazolamide to human erythrocytes was studied in vitro. All experiments were carried out at physiological temperature (37 C) and pH (7.4). Methazolamide (MTZ) buffer concentrations were analyzed by HPLC. Distributional equilibrium between buffer and washed red blood cells was achieved after 1 hr. Results of equilibrium studies were consistent with two classes of binding sites for MTZ within the erythrocyte: a low affinity, high capacity site (CA-I) and a high affinity, low capacity site (CA-II). A two-binding site model was fitted to experimental data generating estimates for binding parameters Ka1 (0.0017 +/- 0.00022 microM-1) nM1 (636 +/- 5.23 microM), Ka2(0.46 +/- 0.0083 microM-1), and nM2(80.9 +/- 0.389 microM). Based upon these findings, kinetic studies were performed in order to characterize the rate of drug distribution. The rate of erythrocyte uptake of MTZ was mathematically modeled using a series of differential equations describing drug diffusion across the red blood cell membrane and subsequent complexation with intracellular binding sites. The model assumed that penetration of MTZ into the red blood cells was passive but drug binding to the carbonic anhydrase isozymes was not instantaneous. Using a novel curve fitting technique, parameter estimates of RBC membrane permeability (0.0102 +/- 0.000618 cm/min), and binding rate constants k-1(0.254 +/- 0.0213 min-1), k1 (0.0022 +/- 0.00020 ml/microgram-min), k-2(1.59 +/- 0.0358 min-1), and k2(3.1 +/- 0.035 ml/microgram-min) were obtained. The model characterized the observed biphasic decline of MTZ buffer concentrations over time and may help explain the prolonged residence of MTZ in vivo.

Blood disposition and urinary excretion kinetics of methazolamide following oral administration to human subjects.

The pharmacokinetic disposition of methazolamide (MTZ) was studied in five healthy volunteers following administration of a single oral dose. Drug concentrations in blood, plasma, and urine were measured by HPLC. Over the range of plasma concentrations observed in vivo, MTZ free fraction (measured by ultrafiltration) was 0.28. Being a carbonic anhydrase inhibitor, MTZ would be expected to distribute into, and be sequestered by, red blood cells. For this reason, MTZ disposition was characterized utilizing blood concentrations as the reference. Using a two-compartment model, a series of differential equations were simultaneously fitted to blood concentrations and urinary excretion data generating estimates for k10 (0.035 +/- 0.019 h(-1)), k12 (0.200 +/- 0.036 h(-1)), k21 (0.077 +/- 0.046 h(-1)), k(a) (0.304 +/- 0.064 h(-1)), Vc (1.1 +/- 0.18 L) and f(r) (fraction excreted renally, 0.61 +/- 0.14). Total blood clearance was 0.037 +/- 0.020 L h(-1). The model estimate of elimination half-life (126 +/- 61 h) was consistent with drug binding to a high affinity carbonic anhydrase isozyme in the erythocyte. Estimates of MTZ renal clearance and renal excretion ratio were 0.021 +/- 0.010 L h(-1) and 0.16 +/- 0.06, respectively. Overall, the prolonged elimination of MTZ from the blood is the result of extensive erythrocyte distribution and tubular reabsorption by the kidney.

Colour vision and side-effects during treatment with methazolamide.

The retina contains Na+K(+)-ATPase and carbonic anhydrase (CA), enzymes that regulate ion fluxes across cell membranes of photoreceptors. Since inhibition of retinal Na+K(+)-ATPase by digitalis impairs colour vision, we wanted to find out whether this also occurs after inhibition of CA. In a double-masked cross-over study with placebo, 14 male volunteers were given 50 mg q.i.d. of the CA inhibitor methazolamide for 2 weeks. A disturbance of colour discrimination was observed in 8 of the 14 subjects, in the classification phase of Lanthony New Color Test. The presence of the disturbance was not significantly correlated to the degree of acidosis or to other side-effects. Its mechanism could be interpreted as a specific effect of CA inhibition in the retina (or the visual cortex) calculated to more than 99.9%.

Time course and disposition of methazolamide in human plasma and red blood cells.

Methazolamide was determined in plasma, whole blood, and urine by a GLC-mass spectrometric method. Temporal patterns of methazolamide concentrations in plasma and red blood cells were obtained following single- and multiple-dose oral administration of the drug. The nonlinearity in the binding of the drug to the red blood cell carbonic anhydrase was evident from a comparison of plasma and red blood cells concentrations. The drug was cleared slowly from the red blood cells. The binding constants to the two isoenzymes of carbonic anhydrase were determined from the plasma and red blood cell concentrations and were in agreement with those determined by previous measurements. The half-life of elimination was 7.5 hr. The urinary recovery of unchanged drug was approximately 25% of the administered dose.

Low-dose methazolamide and intraocular pressure.

Sixteen patients with increased intraocular pressure (over 20 mm Hg) received 25 and 50 mg of oral methazolamide, twice daily, during consecutive weeks and then 500 mg (Sequels) of acetazolamide. The two methazolamide regimens produced significant decreases in intraocular pressure. Acetazolamide treatment resulted in a greater decrease in intraocular pressure but more systemic acidosis and side effects.