Barbara B. Kitchell

Lidocaine plasma protein binding

The percent of unbound lidocaine in the plasma of 24 healthy subjects was measured by equilibrium dialysis after addition of 3 µg/mlC14 lidocaine hydrochloride. The percentage of unbound lidocaine varied from 19.9 to 38.8 (30.2 ± 5, mean ± SD) and was inversely related to the concentration of α1–acid glycoprotein (AAG) in the plasma (r = −0.931, p < 0.001). The binding ratio (number of moles bound divided by number of moles unbound) of lidocaine was directly related to the plasma AAG concentration (r = 0.960, p < 0.001). The binding ratio of lidocaine in solutions containing AAG but no albumin, prepared from the plasma of subjects in the study, was also directly related to the concentration of this acute‐phase protein (r=0.909, p < 0.001). Human serum albumin solution (4 gm/100 ml) bound lidocaine to the extent of 20% under the same conditions. There was no relationship between the binding ratio of lidocaine and the albumin concentration in the plasma of the 24 subjects, in 7 normal subjects variation in AAG between 2 samples collected at least 2 mo apart was associated with a concomitant change in plasma lidocaine binding (r = 0.943, p < 0.01). Thus even in normal subjects there is considerable interindividual and intraindividual variation in lidocaine binding, and measurements of AAG concentration in plasma may be a useful predictor of the extent of lidocaine plasma binding.

Heparin kinetics determined by three assay methods.

Heparin kinetics were determined in four normal subjects, each of whom received three different doses (25, 50, and 75 units/kg body weight) by intravenous injection. Multiple blood samples were collected after each dose and each plasma sample was assayed for heparin activity using three different assay methods. Two of these assays are based on coagulation tests, i.e., activated partial thromboplastin time and thrombin time, while the third is based on chemical neutralization of heparin using hexadimethrine bromide. Heparin kinetics showed pronounced dose‐dependent changes, irrespective of the assay method used, which were characterized by increasing biologic half‐life and decreasing total clearance (Cl) with increasing dose. No changes were noted in apparent volume of distribution (Vd). This data also showed that there were differences in kinetic parameters of heparin depending on the assay method. In general, values for total Cl and apparent Vd based on chemical neutralization were approximately 1.5 to 2.0 times these parameters based on coagulation tests. We conclude that the immediate mechanism of the dose‐dependent heparin kinetics is decreasing total clearance with increasing dose and suggest that in vivo activation of the anticoagulant properties of heparin may explain the assay‐dependent kinetics.

Diazepam and lidocaine plasma protein binding in renal disease

The plasma protein binding of diazepam and lidocaine was measured in patients with renal disease (those with uremia, nephrotic syndrome, or who had received a transplant) and in age‐and sex‐matched control subjects. Percentage unbound diazepam in plasma was increased over control in all three groups of patients as follows: uremic patients 3.23%, control, 1.64% (P < 0.001), nephrotic patients, 3.55%, control, 1.63% (P < 0.001); and transplant recipients, 2.11%, control 1.50% (P < 0.001). The binding ratio (molar concentration of bound to unbound drug) in the patients was related to albumin concentration (r = 0.609, P < 0.001). Percentage of unbound lidocaine did not differ substantially from control in nephrotic patients (34.2%, control 30.8%), but was reduced in the uremic patients (20.8%, control 30.7%, P < 0.001) and transplant recipients (24.6%, control 33.7%, P < 0.005). These increases were associated with increases in α1‐acid glycoprotein (AAG) concentration (uremic patients 134.9 mg/dl, control 66.3, P < 0.001; transplant recipients 106.5, control 65.6, P < 0.001). The binding ratio of lidocaine was closely related to the AAG concentration in patients (r = 0.933, P < 0.001) and controls (r = 0.719, P < 0.001). Thus, the binding of basic drugs may be increased or decreased in patients with renal disease, depending on the relative contribution of the individual plasma proteins to the total binding and the type of disease.

The ability of vascular tissue to produce prostacyclin decreases with age

The ability of aortae from young and mature swine to produce prostacyclin (PGI2) has been determined. PGI2 was measured as its hydration product, 6-keto-PGF1 alpha and assayed by stable isotope dilution GCMS. There was no significant difference in 6-keto-PGF1 alpha production between intimal strips from young and mature aortae in the basal state. In the presence of saturating concentrations of arachidonic acid, however, intimal strips from young aortae synthesized twice as much 6-keto-PFG1 alpha as did older tissues. Fatty acid compositions of young and mature aortae were virtually identical, making dietary difference an unlikely explanation for the age-related decrease in PGI2 synthesis. Both young and mature vascular tissues produced essentially only PGI2; insignificant amounts of PGE2 and PGF2 alpha were found.

The artifactual nature of heparin‐induced drug protein‐binding alterations

Our purpose was to determine whether the reported alteration of protein drug binding after heparin administration in man was artifactual as a result of continued in vitro activity of triglyceride lipases. The lipoprotein lipase inhibitors protamine (14 mg/ml) and ethylenediaminetetraacetic acid (10 mg/ml) were added to blood samples from 11 healthy subjects before and 15 min after 1000 USP units of intravenous heparin. Heparin elevated total nonesterified fatty acid (NEFA) concentration (P < 0.001) and the free fractions of lidocaine, diazepam, and propranolol (P < 0.001 for all). The presence of the lipase inhibitors diminished the heparin‐induced elevation of NEFA (P < 0.001) and free fractions of lidocaine (P < 0.001) and diazepam (P < 0.001), but these values were still greater than control. The inhibitors reduced propranolol binding in control samples and did not diminish the effects of heparin. The change in NEFA concentrations correlated with the free fraction changes of all three ligands (r = 0.739 to 0.849). These data suggest that the heparin‐induced protein binding changes are, to a large extent, in vitro artifacts.

Diazepam and N‐desmethyldiazepam redistribution after heparin

The effects of heparin, 1,000 U intravenously, on the blood, plasma, and free concentrations of diazepam and its metabolite, N‐desmethyldiazepam, have been investigated 3 hr after oral administration of 10 mg diazepam to 5 normal subjects. The percent free diazepam and N‐desmethyldiazepam increased 15 min after heparin from 1.66 ± 0.35 to 3.99 ± 1.88 (mean ± SD; p < 0.05) in the case of diazepam and from 2.50 ± 0.65 to 5.00 ± 1.96 (p < 0.05) in the case of its metabolite. The actual free concentration of diazepam rose from 3.6 ± 1.04 to 6.9 ± 1.33 ng/ml (p < 0.05) 15 min after heparin while total blood concentration was unchanged (144 ± 54 vs 130 ± 57 ng/ml). Free concentrations of N‐desmethyldiazepam rose from 0.62 ± 0.17 to 1.01 ± 0.34 but the effect, though consistent, was not statistically significant. Blood concentrations did not change (15 ± 3.2 vs 14 ± 3.9 ng/ml). That free drug level rose without a change in blood or total plasma levels suggests that factors other than simple plasma binding displacement are involved in this drug interaction.

Effect of temperature on fluorescence and circular dichroism of Escherichia coli dihydrofolate reductase and its complexes.

Dihydrofolate reductase and its complexes have been studied by fluorescence and circular dichroism. NADPH, trimethoprim, pyrimethamine, or Methotrexate binding causes small changes in the enzyme far ultraviolet CD which possibly arise from alterations in polypeptide backbone of the enzyme; however, their effects on enzyme far ultraviolet CD are also explained as the result of ligand interactions with enzyme aromatic groups. In ternary complexes of the enzyme, fluorescence properties of bound NADPH are surprisingly sensitive to the type of inhibitor bound nearby. The effect of temperature on the enzyme and its complexes is clearly shown by changes in enzyme fluorescence and CD. At temperatures near 45 degrees C, the enzyme undergoes an irreversible denaturation, as shown by major alterations in enzyme far ultraviolet CD and by an increased rate of fluorescence quenching. Binary complexes with NADPH or Methotrexate stabilize the enzyme towards this heat denaturation, whereas bound trimethoprim and pyrimethamine do not. Ternary complexes with NADPH and any of the ligands are more stable than the enzyme itself toward heat denaturation. Fluorescence-temperature and fluorescence polarization studies show that near 30 degrees C the enzyme undergoes a reversible transition that is modified by NADPH or methotrexate.

Optical and hydrodynamic studies of the structure of bacteriophage f2 II. Fluorescence of the capsid

Fluorescence properties of the icosahedral RNA virus bacteriophage f2 and its empty capsid are reported. Emission is dominated by tryptophan with a maximum wavelength of 320 nm for f2 and its empty capsid. In addition to this short wavelength maximum, perturbation and denaturation studies indicate the inaccessibility of the tryptophan residues. However a high degree of thermal quenching and a red shift in fluorescence emission on heating suggest a noncooperative structural transition, not a denaturation, which allows buried tryptophans to become exposed to solvent. Therefore the tryptophan residues may be located between subunits. Fluorescence from tyrosine is detected near 315 nm for both f2 and its empty capsid, and may indicate an unusual tyrosine environment. Sensitization of tryptophan fluorescence by tyrosine absorption and low values of polarization indicate tyrosine leads to tryptophan and tryptophan leads to tryptophan energy transfer. The presence of RNA in f2 decreases the efficiency of these transfer processes, but does not significantly affect the other reported fluorescence properties.