Influence of adsorption and deproteination on potential free thyroxine reference methods.

BACKGROUND: There is a need for consensus concerning reference methods to be used for calibration of commercial free-thyroxine (FT(4)) assays. METHODS: Three different potential reference techniques were investigated for adsorption of T(4) to membrane materials, including any in vitro solid surfaces to which T(4) might adsorb, and for efficient separation of the T(4)-binding proteins from the free hormone fraction. We compared ultrafiltration with different commercial ultrafiltration units, ultrafiltration by dialysis tubing, equilibrium dialysis, and ultracentrifugation. We measured the adsorption to membranes and materials with L-[(125)I]T(4). Separation efficiency was determined by measuring the T(4)-binding protein albumin in the ultrafiltrate and the dialysate as well as in the supernatant from the ultracentrifugation with a double-antibody sandwich ELISA technique. RESULTS: We found a constant relationship between the amount of T(4) adsorbed to the dialysis or ultrafiltration membranes/materials and the initial T(4) concentration in HEPES buffer (protein-free medium). T(4) was considerably less adsorbed from serum than from HEPES buffer (P <0.001). Serum T(4) was less adsorbed upon ultracentrifugation than during dialysis and ultrafiltration (P <0.001). It was difficult to completely separate FT(4) from the binding proteins by ultrafiltration and ultracentrifugation. Separation by ultrafiltration depended on the material used. CONCLUSIONS: No investigated separation technique provides technically and theoretically correct separation of the free fraction of T(4) from the protein-bound fraction. Equilibrium dialysis seems to be the least compromised.     

Binding of Antibiotics to Bovine and Ovine Serum

The degree of binding of 37 antibiotics to bovine and ovine serum, after treatment at therapeutic doses, was determined by equilibrium dialysis and ultrafiltration methods. In general, binding values obtained by the two methods were comparable. The extent of binding varied from 0% for cephaloridine and kanamycin to >95% for novobiocin and fusidic acid. Of the 37 drugs studied, one-fourth were less than 25% bound, one-fourth were more than 75% bound, and the percentage binding of about half of the antibiotics ranged from 25 to 75%. Animal to animal variations in the extent of binding of a particular antibiotic were very small. The capacity of bovine or ovine serum to bind antibiotics was, with a few exceptions, similar to the reported capacity of human serum. At drug concentration ranges usually achieved during therapy, variations in drug levels in serum did not influence the degree of binding except with cephalexin, lincomycin, clindamycin, and chloramphenicol. With these antibiotics, the extent of binding increased two- to sevenfold with the decrease in drug concentration in serum.     

Factors affecting the binding of disopyramide to serum proteins

We investigated the influence of the following factors on the binding of disopyramide to serum proteins: method of drug quantification [enzyme immunoassay (EMIT) compared with liquid chromatography (HPLC)], separation technique (ultrafiltration vs equilibrium dialysis), temperature, pH, and total concentration of disopyramide. EMIT and HPLC measurements of disopyramide in ultrafiltrates prepared from 50 sera agreed well: EMIT = 1.046 HPLC + 0.042, (r = 0.928, SEE = 0.04 mg/L). Free disopyramide concentrations in ultrafiltrates of dialyzed sera were similar to those measured in the corresponding dialysates by the EMIT method for 30 patients' sera: ultrafiltrate of serum retentate = 1.053 dialysate + 0.042. Concentrations of free [14C]disopyramide were little affected by temperature. The concentration of free disopyramide decreased as the pH was increased from 7.0 to 7.8. The concentration of free disopyramide, as determined by ultrafiltration, is strongly and directly related to total drug concentration. In the sera of 50 cardiac patients receiving chronic therapy with disopyramide and with total disopyramide concentrations of 0.5 to 5.8 mg/L, the proportion of free disopyramide ranged from 16 to 54%.     

Valproic acid-associated pancreatitis: report of three cases and a brief review

We describe three patients with seizure disorders in whom pancreatitis or pancreatic injury was probably caused by valproic acid, a widely used anticonvulsant drug. Trivial or no increases of serum amylase (EC 3.2.1.1) but striking increases of serum lipase (EC 3.1.1.3) were common to all patients, as assayed in the Kodak Ektachem. In vitro, valproic acid does not cause any change in serum lipase. In patients with symptoms suggestive of pancreatitis and abnormal values for amylase and (or) lipase, treatment with valproic acid should be discontinued.     

Accumulation of indoxyl sulfate, an inhibitor of drug-binding, in uremic serum as demonstrated by internal-surface reversed-phase liquid chromatography

We quantified indoxyl sulfate in uremic serum by using internal-surface reversed-phase high-performance liquid chromatography. Its concentrations were markedly increased in chronic hemodialysis patients, and were significantly but weakly correlated with the concentrations of creatinine and beta 2-microglobulin in these patients' serum, and with the duration of their hemodialysis treatment. Indoxyl sulfate could not be removed effectively by conventional hemodialysis because of its strong binding to serum albumin. Equilibrium dialysis demonstrated that indoxyl sulfate inhibited the binding of salicylate to albumin, and that 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid inhibited the binding of indoxyl sulfate to albumin. In conclusion, indoxyl sulfate was markedly accumulated in uremic serum, and inhibited drug binding.     

Analytical performance evaluation of a new turbidimetric immunoassay for valproic acid on the ADVIA 1650 analyzer: effect of gross hemolysis and high bilirubin

Valproic acid is an anticonvulsant that requires careful therapeutic drug monitoring. Valproic acid is also used in psychiatric patients. Bayer Diagnostics (Tarrytown, NY) recently marketed a turbidimetric immunoassay for monitoring valproic acid concentrations in serum or plasma using the ADVIA 1650 analyzer. We evaluated the performance of this new assay by comparing it with a widely used fluorescence polarization immunoassay (FPIA) on the AxSYM analyzer (Abbott Laboratories, Abbott Park, IL). The total coefficient of variation (CV) for the low control of this new assay was 6.8% (mean = 30.7, SD = 2.1 microg/mL, n = 44) while the corresponding CVs for the medium and high controls were 3.3% (mean = 81.0, SD = 2.7 microg/mL, n = 44) and 5.9% (mean = 142.9, SD = 8.4 microg/mL, n = 44), respectively. The assay is linear up to a serum valproic acid concentration of 170 microg/mL, and the detection limit is 4.4 microg/mL. We observed an excellent correlation between the FPIA of valproic acid and the turbidimetric assay using specimens from 52 different patients who were receiving valproic acid. Using the valproic acid concentrations obtained by the FPIA as the x-axis, and the corresponding valproic acid concentrations obtained by the turbidimetric assay as the y-axis, we developed the following regression equation: y = 1.03 x+1.55 (r = 0.98). With this new assay, high concentrations of bilirubin (unconjugated 30 mg/dL and conjugated 30 mg/dL) and gross hemolysis (4+, hemoglobin: 1,500 mg/dL) have no effect on measurements of valproic acid concentration. We conclude that the new turbidimetric assay for valproic acid can be used for routine therapeutic drug monitoring of valproic acid in clinical laboratories.     

Ultrafiltration devices tested for use in a free thyroxine assay validated by comparison with equilibrium dialysis

A new ultrafiltration method for measuring serum free thyroxine (FT4) is presented. The method makes use of disposable ultrafiltration devices, which were selected on grounds of their ability to produce protein-free serum ultrafiltrates necessary for accurate determination of FT4. Ultrafiltrate thyroxine was measured by radio-immunoassay. As measured by this method, the mean serum FT4 concentration in reference subjects (n = 17) was 25.4 pmol/l (SD 7.8). Two patient groups were studied: hyperthyroid (n = 20, mean serum FT4 138 pmol/l, SD 69) and hypothyroid (n = 18, mean serum FT4 14.8 pmol/l, SD 6.5). These results were compared with serum FT4 concentrations, as measured by an ultrafiltration method based on the use of dialysis-tubing bags (r = 0.97), and by an equilibrium dialysis method (r = 0.95). The mean level of FT4, as measured by the ultrafiltration methodologies, was about twice as high as that measured by equilibrium dialysis. This new FT4 method is more practical than earlier ultrafiltration methods and, theoretically, analytically more accurate than equilibrium dialysis methods.     

Clinical utility of free drug monitoring.

Most drugs are bound to serum proteins to a various degree. Only unbound or free drug is pharmacologically active. Usually total drug is measured for therapeutic monitoring because there is equilibrium between bound and free drugs, and concentration of free drug can be predicted from total drug concentration. However, under certain conditions this equilibrium is disturbed and the measured free drug concentration can be significantly higher than expected from total drug concentrations, especially for strongly protein-bound drugs. In such case a patient may experience drug toxicity even if the total drug concentration is within the therapeutic range. Conditions like uremia, liver disease and hypoalbuminemia can lead to significant increases in free drug concentration. Therefore, monitoring free phenytoin and free valproic acid is recommended in these patients. Drug-drug interactions can also lead to a disproportionate increase in free drug concentration. One strongly protein-bound drug can significantly displace another strongly protein-bound drug if both drugs share the same binding site. Several over-the-counter pain medications such as salicylate, naproxen, and ibuprofen can cause significant displacement of both phenytoin and valproic acid from albumin binding site. Interestingly, such interactions are absent in uremic patients. Elderly patients may have increased free phenytoin or valproic acid due to hypoalbuminemia. Elevated free phenytoin concentrations have also been reported in patients with AIDS. Although digoxin is 25% bound to protein, monitoring free digoxin is useful in patients with elevated endogenous digoxin-like immunoreactive substances or in patients overdosed with digoxin and being treated with digibind. Monitoring free digoxin can also eliminate interference of Chinese medicines Chan Su and Danshen in serum digoxin measurement by certain immunoassays. However, free drug monitoring is not a routine procedure in clinical laboratories due to technical difficulties and lack of established reference ranges for free drugs.     

Plasma protein binding kinetics of valproic acid over a broad dosage range: therapeutic implications

The aim of the study was to characterize, from the relationship between total and free serum levels of valproic acid obtained over a broad dosage range (10–50 mg/kg), the parameters defining the in–vivo kinetic behaviour of the binding of valproic acid to plasma proteins, their pharmacokinetic and clinical repercussions, and their application to therapeutic drug monitoring (TDM). The study was performed in nine healthy adults (20–35 years) who were given doses of 1000 (group A), 2000 (group B) and 3000 mg (group C) of sodium valproate according to a compensated cross–over design, simultaneously determining the total and free serum levels of valproic add over a 24–h period. The mean free fraction increases with dose, although this increase is only significant (P <0 05) for the highest dose (3000 mg). The variation in the free fraction of valproic acid begins to become significant (P < 0>05) at a total drug concentration above 100 mg/1. The mean values of the dissociation constant (K) and binding sites (n) were 460 μmol/l and 179, respectively, showing a variability of 866 and 387%, respectively, and a residual variability of 130%. Significant differences (P < 005) were found for the total plasma clearance (Cl) but not for the intrinsic plasma clearance (CI_u) values, despite their tendency to decrease with the dose. If TDM is to be used for valproic acid, it is the free serum levels that should be determined, especially if high doses are administered, because the total serum levels are not a true reflection of the free ones, as is the case of other anti–epileptic drugs.     

Serum protein-binding characteristics of vancomycin.

A synthesis of studies of serum protein binding of vancomycin and its reported abnormal binding in serum with very high concentrations of immunoglobulin A (IgA) suggests that this antibiotic may be bound to more than one serum protein. Using an ultrafiltration method for separating free from bound drug and high-performance liquid chromatography to measure drug concentration, we studied the binding characteristics of vancomycin for alpha-1 acid glycoprotein, IgG, IgM, IgA, and albumin. The results showed that vancomycin does not bind to alpha-1 acid glycoprotein, IgG, or IgM. Major binding to albumin and IgA occurs, and total drug binding to serum proteins can be fully explained by binding to these two proteins. We calculated an N (number of binding sites per molecule) of 1.3 +/- 0.4 and a K (association constant) of 3.3 x 10(5) +/- 6.3 x 10(4) M-1 (NK = 4.3 x 10(5) M-1) for binding to IgA, whereas the corresponding NK value for albumin was only 527.5 M-1, indicating that vancomycin preferentially binds to IgA. Very high concentrations of IgA in serum (i.e., grams per deciliter), such as in patients with IgA myeloma, may result in the paradox of high (total) concentrations of vancomycin in serum that may be clinically ineffective.     

Free serum cortisol: quantification applying equilibrium dialysis or ultrafiltration and an automated immunoassay system.

BACKGROUND: Quantification of bioactive, free serum cortisol concentrations can characterize adrenocortical function more appropriately compared to total serum cortisol measurement. Ultrafiltration or equilibrium dialysis of serum samples allow direct measurement of free serum cortisol concentrations but respective methods have poorly been validated so far. The aim of our study was to investigate the analytical performance of free serum cortisol measurement employing equilibrium dialysis and ultrafiltration. METHODS: Two commercially available ultrafiltration devices and self-assembled dialysis cells, respectively, were studied. Cortisol was quantified in filtrate or dialysate using an automated immunoassay system. Using two serum pools, the inter-assay coefficient of variation was determined for the three methods and a method comparison was performed. RESULTS: Inter-assay coefficients of variation (n=10) between 3.2% and 14.8% were observed in the imprecision study. Method comparison demonstrated close agreement between free serum cortisol results obtained by ultrafiltration and equilibrium dialysis, respectively (equilibrium dialysis=1.2xultrafiltration+3.9 nmol/L; r=0.99; n=35). CONCLUSIONS: Direct quantification of free serum cortisol after equilibrium dialysis or ultrafiltration of the samples offers acceptable reproducibility and results in close agreement can be obtained. Both methods can potentially be introduced into a routine laboratory setting.     

Ultrafiltration devices tested for use in a free thyroxine assay validated by comparison with equilibrium dialysis

A new ultrafiltration method for measuring serum free thyroxine (FT₄) is presented. The method makes use of disposable ultrafiltration devices, which were selected on grounds of their ability to produce protein-free serum ultrafiltrates necessary for accurate determination of FT₄.

Ultrafiltrate thyroxine was measured by radio-immunoassay. As measured by this method, the mean serum FT₄ concentration in reference subjects (n = 17) was 25.4 pmol/1 (SD 7.8). Two patient groups were studied: hyperthyroid (n=20, mean serum FT₄ 138 pmol/1, SD 69) and hypothyroid (n = 18, mean serum FT₄ 14.8 pmol/1, SD 6.5). These results were compared with serum FT₄ concentrations, as measured by an ultrafiltration method based on the use of dialysis-tubing bags (r=0.97), and by an equilibrium dialysis method (r=0.95). The mean level of FT₄, as measured by the ultrafiltration methodologies, was about twice as high as that measured by equilibrium dialysis. This new FT₄ method is more practical than earlier ultrafiltration methods and, theoretically, analytically more accurate than equilibrium dialysis methods.     

Neurobehavioral effects of phenytoin, carbamazepine, and valproic acid: implications for use in traumatic brain injury

Due to the risk of posttraumatic epilepsy, phenytoin, carbamazepine, and valproic acid are often prescribed for patients with traumatic brain injury (TBI). In this review the literature is examined for evidence of neurobehavioral impairment due to carbamazepine, phenytoin, and valproic acid. No comparative studies have been performed in the TBI population, making if difficult to determine if one of these medications is preferable. Direct inference from studies on epilepsy patients to TBI patients is hazardous due to underlying differences in the two populations. Reported findings for epilepsy patients are subtle and not consistent across studies. All three drugs appear to exert some effect on cognitive and motor functions in epileptic patients, and these impairments worsen at increasing serum levels. The varied length of experience with each drug makes it difficult to assign relative weight to the evidence for or against each. A comparative assessment of cognitive and behavioral effects of anticonvulsants should be done in the TBI population.     

Valproic acid is effective in migraine prophylaxis at low serum levels: a prospective open-label study

OBJECTIVE: We evaluated the efficacy of prophylactic valproic acid treatment (6 months) on the frequency of migraine attacks and the number of migraine headache days with respect to serum levels. BACKGROUND: Valproic acid, a GABAergic drug, has been shown to be effective for migraine prophylaxis. Results from several dose- and serum level-adjusted studies have recommended valproic acid doses within a range of 500 to 1500 mg per day for migraine prophylaxis. Design and METHODS: In this prospective open-label study, 52 patients received valproic acid doses of 300 to 1200 mg per day; 45 patients were treated per protocol. Valproic acid serum levels increased linearly in relation to the valproic acid dose and were between 21 and 107 microg/mL at the end of the treatment period. Patients were divided into two groups: those with valproic acid serum levels less than 50 microg/mL (group 1) and those with serum levels greater than 50 microg/mL (group 2). RESULTS: The frequency of migraine attacks was significantly reduced in group 1 from 3.5 +/- 0.9 to 2.0 +/- 0.9 attacks per month. Migraine headache days also decreased (6.4 +/- 3.5 to 4.6 +/- 2.9 days per month). In the high serum level group, a reduction of migraine attacks from 3.5 +/- 0.9 to 2.8 +/- 1.0 attacks per month and only a slight decrease in headache days (6.4 +/- 3.5 to 6.1 +/- 2.4 days per month) was observed. The outcome of group 1 (low serum level) was significantly better than that of group 2 with respect to both parameters (P<.05). Side effects were generally mild and temporary. CONCLUSIONS: Due to the lack of additional benefit from higher valproic acid doses (more than 600 mg per day), we recommend daily valproic acid doses of 500 to 600 mg with a target serum level less than 50 microg/mL for the prophylactic treatment of migraine.     

Quantifying spurious free T4 results attributable to thyroxine-binding proteins in serum dialysates and ultrafiltrates

BACKGROUND: Direct equilibrium dialysis and direct ultrafiltration free thyroxine (T(4)) assays rely on semipermeable membranes to exclude T(4)-binding serum proteins from dialysates and ultrafiltrates. The presence of these proteins in dialysates or ultrafiltrates will yield spuriously high free T(4) values when free T(4) is quantified by RIA. METHODS: We used a nonanalog free T(4) RIA that detects and quantifies dialyzable and ultrafilterable serum free T(4) to detect T(4)-binding serum proteins. Two equilibrium dialysis devices and 3 ultrafiltration devices were used to illustrate this application. Displacements of [(125)I]T(4) from anti-T(4) by various concentrations of T(4)-depleted thyroxine-binding globulin, albumin, and serum total protein were compared to displacements by various concentrations of free T(4). RESULTS: Both dialysis devices excluded detectable T(4)-binding serum proteins from dialysates. Two of 3 ultrafiltration devices excluded detectable T(4)-binding serum proteins from ultrafiltrates. One did not, and its ultrafiltrate yielded spurious free T(4) values that correlated directly with serum protein concentrations. CONCLUSION: The presence or absence of T(4)-binding proteins in dialysates and ultrafiltrates and the spurious free T(4) values that these proteins cause can be documented using a nonanalog free T(4) RIA.     

Behavioral toxicity of chronic ethosuximide and sodium valproate treatment in the epileptic baboon, Papio papio

The purpose of this study was to assess the effects of chronic administration of gradually increasing doses of the anti-petit mal agents, ethosuximide (15-60 mg/kg/day) and valproic acid (7.5-240 mg/kg/day) on the performance of incremental repeated acquisition and incremental fixed-ratio tasks in the epileptic baboon, Papio papio. At approximately equipotent anticonvulsant doses, ethosuximide (45-60 mg/kg/day) was more behaviorally toxic than valproic acid (7.5-60 mg/kg/day), as determined by the ability of each drug to suppress the incremental repeated acquisition of behavioral chains. The behavioral deficits induced by ethosuximide (60 mg/kg/day) were still present 8 weeks after cessation of drug treatment in two of four animals. Evidence of enhanced acquisition of behavioral chains (decreases in errors) was noted in two of six animals during chronic treatment with valproic acid at a dose of 60 mg/kg/day but was never seen during chronic treatment with ethosuximide. Responding under an incremental fixed-ratio schedule of reinforcement was only affected minimally by either drug. These data suggest that 1) cognitive processes are more vulnerable to disruption by chronic ethosuximide administration than they are to chronic valproic acid administration, 2) performance under the more complex incremental repeated acquisition schedule is more sensitive to disruption than that under an incremental fixed-ratio schedule and 3) the effects of ethosuximide and valproic acid on performance under the incremental repeated acquisition schedule are not due to decreases in motivation or ability to manipulate the response levers.     

Synergistic anticonvulsant effect of valproic acid and ethosuximide on pentylenetetrazole-induced epileptic phenomena in rats

The possible synergistic effect of valproic acid and ethosuximide in combination on pentylenetetrazole-induced epilepsy was investigated in rats. Valproic acid and ethosuximide administered intraperitoneally both showed dose-dependent anti-epileptic activity towards pentylenetetrazole-induced myoclonias and tonic-clonic seizures. The valproic acid-ethosuximide combination had a synergistic pharmacological effect. Against myoclonias combined valproic acid-ethosuximide produced a non-significant decrease in the effective dose of both drugs compared with treatment with either drug alone. In the case of tonic-clonic seizures the protective effect against the seizures was significantly increased by combined treatment compared with treatment with either drug alone. Neither plasma concentrations nor any other pharmacokinetic parameters were significantly changed when the same doses of valproic acid and ethosuximide were given, singly or in combination.     

New ultrafiltration method for free thyroxin compared with equilibrium dialysis in patients with thyroid dysfunction and nonthyroidal illness

This new ultrafiltration method for free thyroxin in serum [FT4(U)] is based on radioimmunoassay of the free hormone fraction in ultrafiltrates obtained by centrifuging serum samples in Unisep Ultracent-10 ultrafiltration devices. We compared the results obtained with those by an equilibrium dialysis method [FT4(D)]. In 36 euthyroid healthy subjects, the mean FT4(U) concentration was 24.2 pmol/L and the mean FT4(D) concentration 14.8 pmol/L. In hyperthyroid and hypothyroid patients, results by the ultrafiltration method were also approximately twice as high as those obtained by the dialysis method. In 23 patients with various nonthyroidal illnesses, mean FT4(U) was 41.2 pmol/L and mean FT4(D) 19.8 pmol/L. The mean FT4(U)/FT4(D) ratio in patients with nonthyroidal illnesses (1.97) was not significantly higher than in control subjects (1.68), making it unlikely that the increase in serum FT4 is caused by weakly protein-bound and therefore dialyzable inhibitors of thyroxin binding to carrier proteins. However, two nonthyroidally ill patients with a clearly increased FT4(U) but a normal FT4(D) concentration might have had such inhibitors, whereas for two other nonthyroidally ill patients a high molar ratio of free fatty acids to albumin is a more likely explanation for increased FT4(U) and FT4(D) concentrations. On theoretical grounds, we consider the FT4(U) concentrations analytically more nearly accurate than FT4(D) values for all patient groups studied.     

Techniques for plasma protein binding of demethylchlorimipramine.

The cerebrospinal fluid (CSF) and plasma levels of demethylchlorimipramine (DMCI) were determined during treatment of depression or obsessive-compulsive disorders with chlorimipramine. In 18 patients the mean CSF/plasma ratio of DMCI was 2.6% +/- 0.7 SD with fourfold variation (1.1% to 4.0%). In spite of this variation, the levels in CSF and plasma at steady state correlated closely (r = 0.91; p less than 0.001). With equilibrium dialysis for the determination of the protein binding of DMCI, a much higher free fraction was found in patients (8.0 +/- 1.6%) and in control subjects (8.2 +/- 1.4%). It was shown that part of the plasma binding capacity was lost during the incubation. Results obtained by ultrafiltration (3.9 +/- 1.0% unbound drug) were closer to the in vivo results, but this method also had disadvantages; much of the drug was absorbed on the ultrafiltration dialysis membrane. Our results suggest that there is a need for care in the selection of a technique for studies of drug protein binding.