Particularly stable elements of noncovalent structure in bovine carbonic anhydrase have been detected and studied. These are present in a highly populated intermediate state formed during denaturation of the enzyme with guanidinium chloride. The intermediate has been detected by analysis of the denaturation profiles, and some of its structural properties have been characterized by CD and fluorescence spectroscopy, including fluorescence polarization and lifetime measurements. Measurements have been made on the Zn2+-enzyme, Co2+-enzyme, and apoenzyme to ascertain the structural effects of the active-site Zn2+. Kinetic measurements indicate that this intermediate is on the folding pathway from the random coil to the native state.

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.

The percent of unbound lidocaine in the plasma of 24 healthy subjects was measured by equilibrium dialysis after addition of 3 microgram/ml C14 lidocaine hydrochloride. The percentage of unbound lidocaine varied from 19.9 to 38.8 (30.2 +/- 5, mean +/- SD) was inversely related to the concentration of alpha 1-acid glycoprotein (AAG) in the plasma (r = -0.931, p less than 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 less than 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 less than 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 1 mo apart was associated with a concomitant change in plasma lidocaine binding (r = 0.943, p less than 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.

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 less than 0.001), nephrotic patients, 3.55%, control, 1.63% (P less than 0.001); and transplant recipients, 2.11%, control 1.50% (P less than 0.001). The binding ratio (molar concentration of bound to unbound drug) in patients was related to albumin concentration (r = 0.609, P less than 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 less than 0.001) and transplant recipients (24.6%, control 33.7%, P less than 0.005). These increases were associated with increases in alpha 1-acid glycoprotein (AAG) concentration (uremic patients 134.9 mg/dl, control 66.3, P less than 0.001; transplant recipients 106.5, control 65.6, P less than 0.001). The binding ratio of lidocaine was closely related to the AAG concentration in patients (r = 0.933, P less than 0.001) and controls (r = 0.719, P less than 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 to the total binding and the type of disease.