And Alpha 1-acid Glycoprotein Research Articles

Factors influencing the protein binding of a new phosphodiesterase V inhibitor, DA‐8159, using an equilibrium dialysis technique

Various factors influencing the protein binding of DA-8159 to 4% human serum albumin (HSA) were evaluated using an equilibrium dialysis technique at an initial DA-8159 concentration of 5 microg/mL. It took approximately 8 h incubation to reach an equilibrium between 4% HSA and an isotonic phosphate buffer of pH 7.4 containing 3% of dextran ('the buffer') using a Spectra/Por 2 membrane (mol. wt. cut-off: 12,000--14,000) in a water bath shaker kept at 37 degrees C and at a rate of 50 oscillations per min. The extent of binding was dependent on DA-8159 concentrations, HSA concentrations, incubation temperature, buffer pH, and alpha-1-acid glycoprotein (AAG) concentrations. The binding of DA-8159 in heparinized human plasma (93.9%) was significantly higher than in rats (81.4%), rabbits (80.4%), and dogs (82.2%), and this could be due to differences in AAG concentrations in plasma.

Serum protein binding of tolterodine and its major metabolites in humans and several animal species.

The aim of this study was to determine in vitro protein binding of tolterodine and its 5-hydroxymethyl (5-HM) and N-dealkylated metabolites in serum from humans and several animal species at concentrations similar to those obtained in clinical and preclinical studies. Binding of tolterodine and the two metabolites to human serum albumin and alpha1-acid glycoprotein (AAG) was also assessed, as was binding of tolterodine to red blood cells. Ex vivo protein binding of tolterodine and 5-HM was determined in serum samples from healthy volunteers treated with oral tolterodine 4 mg twice daily for 8 days. Tolterodine exhibited high protein binding in human serum; the unbound fraction (f(u)) was 3.7%. The unbound fraction of tolterodine in cat and dog serum (1.5 and 2.1%, respectively) was lower compared with human serum; f(u) was higher in the other species investigated (rat, 22%; mouse, 16-17%; rabbit, 39%). The unbound fraction of 5-HM was much higher in serum from humans (36%) and all animal species investigated (mouse, 72%; rabbit, 68%; cat, 32%; dog, 45%). Binding of N-dealkylated tolterodine to proteins in human serum was intermediate (f(u) 14%). AAG was the major binding protein for tolterodine and 5-HM, and the degree of binding increased with increasing concentration of the protein. The association constant of 5-HM for AAG was lower than that of tolterodine (1.3 x 10(5) M(-1) versus 2.1 x 10(6) M(-1)). The blood:plasma tolterodine concentration ratio was 0.6 in both humans and dog; thus, a minor fraction of tolterodine was present in red blood cells compared with plasma (0.18 and 0.36, respectively). In the mouse, tolterodine was equally present in blood and plasma. In ex vivo samples, f(u) values for tolterodine (pH adjusted) varied between 1.6 and 4.9% (mean 2.8%), which could be explained by differences in AAG concentrations. There was good correlation between observed f(u) values for tolterodine and those predicted on the basis of AAG levels. Similar findings were observed for 5-HM.

Pharmacokinetics and Efficacy of Long-Term Epidural Ropivacaine Infusion for Postoperative Analgesia

The aim of this study was to evaluate the pharmacokinetics and efficacy of the new local anesthetic ropivacaine when used for epidural infusion for up to 72 h after major orthopedic surgery. Immediately after surgery, an epidural infusion of ropivacaine 2 mg/mL was begun at a rate of 6 mL/h in 11 patients. The infusion rate was then adjusted according to patient analgesic needs or side effects. Blood samples were taken during and after the infusion to determine total and unbound ropivacaine and alpha1-acid glycoprotein (AAG) concentrations. Patients were assessed regularly for sensory and motor block and pain using a visual analog scale (VAS) score (0-100 mm). Ten patients received 63-72 h of infusion. Total plasma concentrations of ropivacaine and binding protein (AAG) increased during the infusion such that free concentrations plateaued or began to fall over time. VAS values during mobilization were less than 40 mm in 93% of patients. The majority of patients had no measurable motor block once the surgical block had regressed. When epidural ropivacaine was titrated to achieve a stable sensory block, there was a low incidence of motor block, and free plasma ropivacaine levels were well below the toxic range. The pharmacokinetics of continuous epidural infusions of ropivacaine are described in patients for up to 72 h postoperatively. Clinical efficacy and side effects are also reported. An understanding of the plasma concentrations obtained and modes of elimination during prolonged epidural infusion is important for safe, routine clinical use in postoperative analgesia.

Pharmacokinetics and Efficacy of Long-Term Epidural Ropivacaine Infusion for Postoperative Analgesia

The aim of this study was to evaluate the pharmacokinetics and efficacy of the new local anesthetic ropivacaine when used for epidural infusion for up to 72 h after major orthopedic surgery.Immediately after surgery, an epidural infusion of ropivacaine 2 mg/mL was begun at a rate of 6 mL/h in 11 patients. The infusion rate was then adjusted according to patient analgesic needs or side effects. Blood samples were taken during and after the infusion to determine total and unbound ropivacaine and alpha1-acid glycoprotein (AAG) concentrations. Patients were assessed regularly for sensory and motor block and pain using a visual analog scale (VAS) score (0-100 mm). Ten patients received 63-72 h of infusion. Total plasma concentrations of ropivacaine and binding protein (AAG) increased during the infusion such that free concentrations plateaued or began to fall over time. VAS values during mobilization were less than 40 mm in 93% of patients. The majority of patients had no measurable motor block once the surgical block had regressed. When epidural ropivacaine was titrated to achieve a stable sensory block, there was a low incidence of motor block, and free plasma ropivacaine levels were well below the toxic range. Implications: The pharmacokinetics of continuous epidural infusions of ropivacaine are described in patients for up to 72 h postoperatively. Clinical efficacy and side effects are also reported. An understanding of the plasma concentrations obtained and modes of elimination during prolonged epidural infusion is important for safe, routine clinical use in postoperative analgesia. (Anesth Analg 1997;85:1322-30)

Fentanyl and alfentanil plasma protein binding in preterm and term neonates.

The effects of gestational age (GA) and plasma protein concentrations on the plasma protein binding of fentanyl and alfentanil were studied in preterm and term neonates. Binding experiments were performed using split-cell equilibrium dialysis. Fentanyl and alfentanil concentrations were measured using specific radioimmunoassay, and the proteins albumin and alpha-1-acid glycoprotein (AAG) were measured using radial immunodiffusion assays. In the preterm neonates, 77% of fentanyl and 65% of alfentanil was bound. In the term neonates, 70% of fentanyl and 79% of alfentanil was bound. The binding ratio of alfentanil showed a positive correlation with gestational age and AAG concentration. The binding ratio of fentanyl showed a weak, negative correlation with gestational age. These data indicate that fentanyl and alfentanil are not interchangeable at the GA studied because of age-related changes in protein binding.

C-reactive protein and alpha 1-acid glycoprotein levels in dogs infected with Ehrlichia canis

To elucidate whether acute-phase protein responses occur in dogs infected with Ehrlichia canis, C-reactive protein (CRP) and alpha 1-acid glycoprotein (AAG) levels were serially measured in the plasma of five dogs experimentally inoculated with E. canis and 10 sham-inoculated or noninoculated control dogs. The CRP concentration was measured by a canine-specific capture enzyme-linked immunosorbent assay, and the AAG concentration was measured by a canine-specific radial immunodiffusion method. In all E. canis-inoculated dogs, a 3.3- to 6.5-fold increase in the plasma CRP concentration and a 1.9- to 8.6-fold increase in the plasma AAG concentration over the preinoculation level occurred at days 4 to 6 postexposure. Despite the persistence of E. canis and high antibody titers, both CRP and AAG concentrations gradually declined to preexposure levels by day 34 postexposure. E. canis-infected dogs had mild and transient clinical signs which resolved without treatment by day 14 postexposure. The CRP and AAG concentrations in control inoculated or nontreated dogs remained within the normal range throughout the experimental period. Of 12 dogs naturally infected with E. canis, 75% had greater than 50 micrograms of CRP per ml and 83% had greater than 500 micrograms of AAG per ml. All of these 12 dogs had chronic and severe clinical signs of canine ehrlichiosis. Thus, elevations in the levels of acute-phase proteins occur in both acute and chronic canine ehrlichiosis. Determination of CRP and AAG concentrations may help in assessing the severity of inflammatory damage in dogs with E. canis infections.

Concanavalin A-bound selenoprotein in human serum analyzed by graphite furnace atomic absorption spectrometry

We developed an assay for the direct determination of selenium in serum with a Perkin-Elmer Model 4100ZL Zeeman atomic absorption spectrometer and Ag-Cu-Mg modifier. We used this assay to analyze concanavalin A-bound selenoprotein (CABSP) in human serum after concanavalin A (ConA) affinity chromatography. The CABSP was identified as a single-chain glycoprotein of 57.3 kDa. Carbohydrate units were N- and O-linked to the protein. The selenium moiety was selenocysteine. Total selenium, glutathione peroxidase (GPX; EC 1.11.1.9), ConA-bound selenium (CABS), and alpha 1-acid glycoprotein (AAG) were determined in normal subjects and patients with various pathological conditions. CABS accounted for 44.1% +/- 6.3% of total selenium in sera from normal subjects and 46.5% +/- 3.9% to 55.1% +/- 8.1% in sera from patients with a variety of diseases. Total selenium in serum was well correlated with serum CABS (r = 0.860), but not with serum GPX activity (r = 0.117), for all patients studied. Serum CABS increased in normal subjects after selenium supplementation. Serum CABSP did not behave as an acute-phase reactant, compared with AAG.

Influence of Age and Gender on the In Vitro Serum Protein Binding of Flecainide

The in vitro protein binding of flecainide was studied by equilibrium dialysis in relation to serum concentrations of albumin and alpha 1-acid glycoprotein (AAG) in 22 healthy subjects of both sexes aged between 23 and 89 years. In the range of flecainide concentrations tested, protein binding of flecainide was not saturable and the percent value of the unbound fraction ranged between 0.48 and 0.68, mean value (SD)=0.59 (±0.06), without any significant difference between males and females or between young and old subjects. The flecainide unbound fraction was significantly correlated with serum albumin concentrations but not with total serum proteins or AAG concentrations.

Accumulation of a Disopyramide Metabolite in Renal Failure

Ten hemodialysis (HD) patients with normal liver function received disopyramide (DP) therapy in the steady state. DP, mono-N-dealkyldisopyramide (MND) and alpha-1-acid glycoprotein (AAG) were measured before and after HD. Ten patients with normal renal and liver function were selected as controls. The DP concentration was 2.08 +/- 0.39 micrograms/ml (mean +/- SD) in the control group, and the pre and post HD levels were 2.40 +/- 1.08 micrograms/ml and 1.73 +/- 0.87 micrograms/ml, respectively, in the HD group. The MND concentration was 0.42 +/- 0.23 microgram/ml in controls, 1.53 +/- 0.52 micrograms/ml in pre HD and 1.08 +/- 0.32 microgram/ml in post-HD. Although DP and MND are both classified as small molecular weight substances, the average decrease in plasma concentration from pre to post HD was under 30% with both agents. The MND/DP ratio in the HD group was higher than in controls, but there was no significant difference between pre and post HD. The AAG level was 75 +/- 5 mg/dl in controls and 109 +/- 11 mg/dL before HD in the HD group (p < 0.001). In conclusion, an MND accumulation was observed in HD patients receiving DP therapy. Because the anticholinergic effect of MND is 24 times that of DP, MND is thought to contribute in some way to the hypoglycemia induced by DP in renal failure.

Plasma Protein Binding of Lidocaine and Warfarin in Insulin-Dependent and Non-Insulin-Dependent Diabetes Mellitus

We examined the plasma protein binding of an acidic drug (warfarin bound to albumin) and a basic drug [lidocaine (lignocaine) bound to alpha 1-acid glycoprotein] in 15 patients with insulin-dependent diabetes mellitus (IDDM) and 15 matched controls. We also examined protein binding of warfarin and lidocaine in 30 patients with non-insulin-dependent diabetes (NIDDM) and 25 controls. Compared with control, the binding of both warfarin (98.81 +/- 0.02 vs 98.57 +/- 0.03%, mean +/- SEM) and of lidocaine (69 +/- 2 vs 58 +/- 2%) was significantly reduced in IDDM. This group had lower concentrations of both albumin and alpha 1-acid glycoprotein (AAG), achieving statistical significance vs control for albumin only. In the patients with NIDDM, who had a similar level of glycosylated haemoglobin, while there was no significant difference in the binding of lidocaine there was a significant increase in warfarin binding compared with the control population (99.01 +/- 0.03 vs 98.82 +/- 0.04%). This study suggests that binding of both acidic and basic drugs is altered in both IDDM and NIDDM.

PLASMA CONCENTRATIONS OF BUPIVACAINE AND TWO OF ITS METABOLITES DURING CONTINUOUS INTERSCALENE BRACHIAL PLEXUS BLOCK

An interscalene brachial plexus block was performed via a catheter with 20-28 ml of 0.75% bupivacaine plus adrenaline for surgery of the shoulder region in 12 patients. Constant infusion of 0.25% bupivacaine 0.25 mg kg-1 h-1 was continued for 24 h. During surgery light general anaesthesia, without analgesics, was maintained. Plasma concentrations of total and unbound (free fraction) bupivacaine, desbutylbupivacaine (DBB), 4-hydroxybupivacaine (4-OHB) and alpha 1-acid glycoprotein (AAG) were measured at predetermined intervals during the continuous block. The greatest mean plasma concentrations of bupivacaine were measured at 30 min (1.63 (SD 0.55) micrograms ml-1) and 60 min (1.38 (0.48) micrograms ml-1). There was a small but statistically significant increase in the plasma concentration of bupivacaine between 12 and 24 h of infusion. The mean unbound concentration of bupivacaine in plasma decreased from 0.044 (0.015) microgram ml-1 (3.6 (1.1)% of total bupivacaine concentration) at 3 h to 0.023 (0.011) micrograms ml-1 (2.1 (1.0)%) at 24 h. The AAG concentration in plasma increased by 38% in 24 h. The metabolites DBB and 4-OHB were detectable in plasma from 30 min, with a gradual increase during infusion. At 24 h the mean concentrations of DBB and 4-OHB were 0.33 (0.22) micrograms ml-1 and 0.13 (0.04) micrograms ml-1, respectively. There were no toxic reactions during the blocks.

The effect of age on serum concentrations of albumin and alpha 1‐acid glycoprotein.

1. Human serum albumin (HSA) concentrations and alpha 1-acid glycoprotein (AAG) concentrations were measured in 68 subjects, 35 males and 33 females, aged 20-90 years without evidence of acute or chronic inflammatory disease or malignancy. Subjects were drug free for at least 1 month. HSA and AAG concentrations were measured using rate nephelometry. 2. Age had no effect on alpha 1-acid glycoprotein concentration, whereas plasma albumin levels decreased as a function of age in both sexes. We observed no differences between males and females in the plasma concentrations of HSA and AAG. 3. These data show that in healthy subjects the HSA concentration decreases with increasing age, whereas age, uncomplicated by disease does not influence AAG concentration.

Use of 1-Anilino-8-naphthalene Sulfonate as a Fluorescent Probe in the Investigation of Drug Interactions with Human Alpha-1-acid Glycoprotein and Serum Albumin

We report a rapid method for the characterization of the human serum albumin (HSA) and alpha-1-acid glycoprotein (AAG) interactions with drugs. The binding of 1-anilino-8-naphthalene sulfonate (ANS) to AAG and HSA was measured by fluorescence spectroscopy. Fluorescence data indicated that ANS was bound tightly to at least one site on AAG, with an affinity constant of 1.35 × 106 M−1. The fluorescence of an ANS:AAG complex was quenched by the binding of various drugs. Fluorescence quenching of the HSA:ANS complex showed a single site with an affinity constant of 0.72 × 106 M−1. The interaction of AAG and HSA with ANS or other drugs was also studied by comparative equilibrium dialysis. [14C]Pipequaline was used as an AAG and HSA site marker. [14C]Pipequaline seems to share sites I (azapropazone) and II (diazepam and ibuprofen) of HSA. However, high concentrations of warfarin were unable to displace [14C]pipequaline. On the other hand, it was shown that palmitic acid decreased, whereas bilirubin increased the pipequaline binding.

Pharmacokinetics of Continuous Intravenous Infusion of Methadone in the Early Post‐Burn Period

The pharmacokinetics of methadone were studied in 14 patients with acute, severe burns and receiving an intravenous infusion of methadone to control their pain. Serum methadone concentrations were measured by gas chromatography on 5 mL arterial blood samples obtained at 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0 and 24 hours after the start of infusion. Albumin and Alpha-1-Acid glycoprotein (AAG) were measured by radial immunodiffusion. Serum methadone concentration-time data were fit with the appropriate sum of exponentials equation using iterative nonlinear regression analysis. All serum methadone concentration-time data were best described by a monoexponential equation. Estimates of Vd (180 +/- 62 L) were not significantly different from those predicted for Vc from body weight using literature values (156 +/- 41). Estimates of Vd were, however, significantly lower than those predicted for Vz using literature values (282 +/- 74) (P less than 0.001). In addition, CL values (53.0 +/- 19.3 L/h) were significantly higher than those predicted from body weight using literature values (9.2 +/- 2.3 L/h) (P less than 0.001). These changes resulted in estimates of the elimination half-life for methadone of 2.6 +/- 1.1 h. Methadone protein binding was independent of both albumin and AAG concentration. Multiple regression demonstrated that the significant predictors of CL in the early post burn injury period were serum albumin, days post injury and age. The coefficient of determination (r2) for this model was 0.8190. In summary, methadone CL is markedly elevated while the Vc is essentially unchanged during the early post burn injury period.(ABSTRACT TRUNCATED AT 250 WORDS)

Possible Effect of Lactational Period on the Milk-to-Plasma Drug Concentration Ratio in Lactating Women: Results of an In Vitro Evaluation

The fat and protein composition of human milk changes dramatically in the first several weeks postpartum. In order to investigate the possible effect of this compositional change on the milk-to-plasma drug concentration ratio (M/P), the following experiment was performed. Milk samples were collected from five healthy lactating women on days 3, 5, 7, and 14 postpartum; blood samples were obtained on these days, as well as on day 1. Serum and skim milk unbound fractions (fp and fm, respectively) and the skim milk-to-whole milk drug concentration ratio (S/M) were determined in vitro in the above samples for diazepam, propranolol, and etretin, an aromatic retinoid. In addition, the composition of these milk and serum samples was also assessed. Using a previously proposed mathematical model for the distribution of drugs between milk and plasma, M/P was calculated from values for fm, fp, S/M, milk pH, and literature values for the pKa values of the compounds. The M/P was calculated for each subject on each day of sample collection. Total serum protein and alpha-1-acid glycoprotein (AAG) levels increased in the first two weeks postpartum. Mean diazepam fp values fell from 0.020 to 0.014 during this period, while propranolol fp values changed inversely with serum AAG levels. Milk whey and total proteins decreased as lactation progressed, but changes in fat levels were not statistically significant. Calculated propranolol M/P increased during the study period, predominately due to changes in milk pH and propranolol S/M.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in lignocaine disposition during long-term infusion in patients with acute ventricular arrhythmias.

Lignocaine disposition was studied in 30 patients with acute ventricular arrhythmias. Serum concentrations of lignocaine, its metabolites Monoethylglycine xylidide (MEGX) and glycine xylidide (GX), and alpha 1-acid glycoprotein (AAG) were analyzed during and after a 48-h lignocaine infusion. AAG concentrations tended to rise in patients with acute myocardial infarction (AMI), leading to binding of the drug in plasma. Lignocaine clearance was estimated at various times during the infusion using a Bayesian parameter estimation program and was found to decline over the course of the infusion. There was a significant reduction in clearance based on estimates obtained at the end of the infusion compared with estimates obtained during the first 0-5 h. Clearance was reduced both in patients who had an AMI and those who did not. Multiple linear regression analysis of the clearance data revealed that these changes could be described by a linear function of time and AAG concentration. These findings suggest that other factors in addition to protein binding changes may influence lignocaine disposition during long-term infusion.

Determinants of carbamazepine and carbamazepine 10,11-epoxide binding to serum protein, albumin and alpha 1-acid glycoprotein.

The binding of carbamazepine and carbamazepine 10,11-epoxide to serum, albumin and alpha 1-acid glycoprotein (AAG) was determined and compared at drug concentrations ranging from 0.5 to 400 mg/l using equilibrium dialysis and liquid chromatography. The total binding of carbamazepine in serum was determined primarily by albumin and to a lesser extent (20-30%) by AAG. Modified Scatchard plots for carbamazepine binding in serum were biphasic, suggesting the presence of two binding sites on serum protein. Association constants characterizing the first (k1 = 2.4 X 10(4) l/mol) and second (k2 = 4.6 X 10(2) l/mol) binding sites agreed with those measured for AAG and albumin respectively. Modified Scatchard plots for carbamazepine 10,11-epoxide binding in serum were linear and serum binding was largely accounted for by binding to albumin. The epoxide metabolite did not bind to AAG. Carbamazepine binding to AAG was drug concentration-dependent over the concentration range considered to be therapeutic, while the percent binding values for carbamazepine and epoxide binding to albumin and serum from a normal individual were constant over this range. Computer simulations showed that physiological extremes in AAG and albumin concentrations can result in a range of carbamazepine unbound fractions of 0.17 to 0.47. These data suggest that normal variations in concentrations of both proteins may be the principal cause of interpatient variability in serum protein binding of carbamazepine.