Free Valproic Acid Research Articles

Solid-phase microextraction for the determination of the free concentration of valproic acid in human plasma by capillary gas chromatography

The potential of solid-phase microextraction in the bioanalysis of drugs is demonstrated. The free concentration of valproic acid in human plasma was determined by equilibrium dialysis followed by solid-phase microextraction and capillary gas chromatography. Human plasma samples were dialysed at room temperature. To the dialysate was added an internal standard and the pH was adjusted to 2.5. The polymethylsiloxane-coated fused-silica fibre of the solid-phase microextraction device was inserted into the dialysate for 3 min. The sorbed analytes were then thermally desorbed at 210°C in the split-splitless injection port of the gas chromatograph, separated on a Nukol capillary column and detected with a flame ionization detector. The method was shown to be highly reproducible with a detection limit of 1 μg/ml of free valproic acid in human plasma.

Valproate-mediated disturbances of hemostasis: relationship to dose and plasma concentration.

Valproic acid (VPA) may cause impaired platelet function, thrombocytopenia and, occasionally, severe bleeding. Controversy exists both as to the mechanism of this alteration in hemostasis and as to whether these adverse effects are either dose-related or idiosyncratic. Previous investigations have focused primarily on pediatric patients who commonly were receiving multiple anticonvulsant medications. We evaluated a cohort of 27 adult patients with epilepsy who were receiving VPA monotherapy and compared them with age-matched controls to determine whether a correlation exists between platelet count, function, bleeding time, levels of von Willebrand's factor antigen, and VPA dose or plasma concentration. VPA patients had significantly lower platelet counts and longer bleeding times than did controls (p < 0.05). Platelet count was inversely correlated to VPA dose and both free and total VPA concentration (p < 0.01). There was no significant difference in levels of von Willebrand's factor antigen between patients and controls. We assessed platelet aggregation by measurement of whole-blood platelet aggregation and release with agonists including adenosine diphosphate, thrombin, collagen, arachidonic acid, and ristocetin. VPA patients had significant decreases in platelet aggregation values compared with controls. There were significant differences in collagen, arachidonic acid, and adenosine diphosphate release and aggregation between groups that correlated to both VPA dose (p < 0.01) and concentration (p < 0.05). Prolongation of bleeding time was significantly correlated to both VPA dose and concentration. Our data suggest a significant relationship between impaired platelet function and both VPA dose and plasma concentration.

Reduced In Vitro Displacement of Valproic Acid From Protein Binding by Salicylate in Uremic Sera Compared with Normal Sera:Role of Uremic Compounds

Limited studies indicate increased free fraction of valproic acid in uremia. One study also described displacement of valproic acid from protein binding by salicylate in children with epilepsy. The authors studied in vitro interaction of valproic acid and salicylate in uremic sera and compared their results with same interactions in normal sera. As expected, the concentrations of free valproic acid were always significantly higher in uremic sera compared with normal sera. When uremic sera were supplemented with both valproic acid and salicylate, a much lower displacement of valproic acid by salicylate was observed, compared with the effects observed in normal sera. Treatment of uremic sera with charcoal removed those uremic compounds, and the concentration of free valproic acid in charcoal-treated uremic sera were comparable to the concentrations observed in normal sera. When uremic compounds were extracted from charcoal with methanol, lyophilized, and added to normal serum, an increase in free valproic acid concentration in normal serum was observed, indicating that uremic compounds, rather than altering albumin structure, are responsible for elevated free valproic acid concentration in uremia. However, uremic extract failed to produce any further displacement of valproic acid in normal serum in the presence of salicylate. The authors concluded that uremic compounds are responsible for elevated free valproic acid concentrations, but the displacement of valproic acid by salicylate in uremic sera are less remarkable compared with such effect in normal sera.

蛍光偏光免疫測定法による血清中遊離形バルプロ酸濃度測定とその動態

蛍光偏光免疫測定法による血清中遊離形バルプロ酸濃度測定とその動態

Simultaneous “In Series” Hemodialysis and Hemoperfusion in the Management of Valproic Acid Overdose

There is little published experience with the extracorporeal treatment of valproic acid overdose, although the pharmacokinetic properties of valproic acid suggest a potential role for hemodialysis and/or hemoperfusion. At therapeutic concentrations valproic acid is highly protein bound, but at the higher concentrations seen in the overdose setting binding sites become saturated and free valproic acid should be subject to removal by hemodialysis. We report the case of a patient with serious toxicity secondary to valproic acid overdose who was successfully treated with a combination of hemodialysis and hemoperfusion. With this treatment the half-life of valproic acid was reduced from a predialysis level of 13 hours to 1.7 hours, with rapid lowering of valproic acid levels and dramatic clinical improvement. Based on our experience in this patient and a review of previously reported cases, hemodialysis should be considered in the treatment of valproic acid overdose.

Determination of Free Valproic Acid

Valproic Acid (VPA) is an important drug for the treatment of several types of seizures because it has a wide spectrum of activity. Since VPA has an unusual nonlinear binding characteristic and a wide interindividual variation, monitoring of its free concentration can be helpful in patient management. The determination of unbound VPA is more difficult because an extra sample preparation step is needed and the concentration of free VPA is low. Free drug monitoring can assume a more important role if there is a refinement in the technology. A high-performance liquid chromatography (HPLC) method with isocratic elution has been established for the analysis of the 4-bromomethyl-7-methoxycoumarin (BrMMC) derivative of free VPA. This method has a better sensitivity, linearity, and precision than enzyme immunoassay (EIA). Ultrafiltration with the Centrifree system was evaluated for the sample preparation. The influence of centrifuge times, relative centrifugal forces, and the starting sample amounts on the final results of the ultrafiltration were investigated. There was a satisfactory correlation between the free VPA levels determined by the HPLC method and the concentrations obtained by EIA. The total and free VPA were determined on 100 samples from 36 patients. The total VPA levels were in a range of 25 to 208 micrograms/ml, free VPA concentrations ranged from 1.92 to 55.75 micrograms/ml with the free fractions from 7 to 37%.

Comparison of anticonvulsant effects of valproic acid entrapped in positively and negatively charged liposomes in amygdaloid-kindled rats

Intraperitoneal injection of free valproic acid (VPA) suppressed amygdaloid-kindled seizure 1 h after injection in rats, but had no effect at 24 h. VPA entrapped in positively charged liposomes showed a prolonged anticonvulsant effect lasting for 2 days, while the effect evaluated at 1 h was not different from that with free VPA. VPA entrapped in negative charged liposomes exerted a significantly stronger effect at 1 h than did free VPA, while it had no significant effect at 24 h. These results suggest that surface charges on liposomes play an important role in modifying the anticonvulsant effect of VPA.

Determination of valproic acid by high-performance liquid chromatography with photodiode-array and fluorescence detection

The derivatization of valproic acid and undecylenic acid with 4-bromomethyl-7-methoxycoumarin is described. The derivatives were detected by photodiode-array and fluorescence detectors. The optimum monitoring conditions and the stability of the suspension solution and derivatives were investigated. A high-performance liquid chromatographic (HPLC) method with isocratic or gradient elution has been established for the analysis. This method has been used for the determination of total and free valproic acid in serum. There is a satisfactory correlation between the results obtained by this HPLC method and those measured by the enzyme immunoassay. Some other common anti-epileptic drugs did not interfere with the analysis. The method is simple and fast and has better sensitivity and linearity than enzyme immunoassay. It is suited for routine therapeutic drug monitoring.

Pharmacokinetics of total and free valproic acid during monotherapy in infants

The pharmacokinetics of free and total valproic acid (VPA) in plasma and whole blood after oral administration during steady state was investigated in seven infants (mean age 10.7 months) receiving monotherapy. The VPA concentrations in whole blood closely followed those in plasma but at a reduced level. A positive correlation was found between dose and mean plasma concentration (r = 0.71). Mean terminal half-lives were similar in plasma and whole blood (12.5 and 15.5 h, respectively), but were considerably longer than for free VPA (6.4 and 6.5 h, respectively; P less than 0.01). There was a significant decrease in half-lives with increasing age (P less than 0.05). Plasma and whole blood clearance for total VPA was higher than reported in older infants and adults (17.8 and 28.9 ml/kg per hour) and was considerably higher for free VPA (127.6 and 188.8 ml/kg per hour, respectively). The increase in clearance compared with that in older subjects is well in concordance with a lower protein binding of VPA (mean 85.3%). Of special importance is that the percentage of unbound VPA increased with increasing concentrations of total VPA. The fraction of unbound VPA in plasma increased even more in subjects with low albumin concentrations (P less than 0.01).

Total and free Valproic acid: plasma level/dose ratio in monotherapy

Total and free Valproic acid: plasma level/dose ratio in monotherapy

The enzymatic basis for the metabolism and inhibitory effects of valproic acid: dehydrogenation of valproyl-CoA by 2-methyl-branched-chain acyl-CoA dehydrogenase

Five distinct acyl-CoA dehydrogenases are currently known. These are short, medium, long and 2-methyl-branched-chain acyl-CoA dehydrogenases, and isovaleryl-CoA dehydrogenase. We tested these five acyl-CoA dehydrogenases for their ability to dehydrogenase valproyl-CoA using pure enzyme preparations isolated from rat liver mitochondria. The activities of the pure human short-chain, medium-chain, and isovaleryl enzymes purified from post-mortem livers, and a long-chain acyl-CoA dehydrogenase preparation partialy purified from placental mitochondria, were also tested. Valproyl-CoA was dehydrogenated at a significant rate (0.167 μmol/min per mg protein) only by rat 2-methyl-branched-chain acyl-CoA dehydrogenase. Human 2-methyl-branched-chain acyl-CoA dehydrogenase has not been purified; therefore, it could not be tested. Since four other human acyl-CoA dehydrogenases did not dehydrogenate isobutyryl-CoA, 2-methylbutyryl-CoA (obligatory intermediates from valine and isoleucine, respectively) nor valproyl-CoA, it is reasonable to assume that valproyl-CoA is dehydrogenated by 2-methyl-branch-chain acyl-CoA dehydrogenase in man as well. We identified 2-propyl-2-pentenoyl-CoA as the reaction product from valproyl-CoA by mass spectral analysis of the acyl moiety. Valproyl-CoA, at 0.3 mM, moderately inhibited human acyl-CoA dehydrogenases with the exception of the long-chain enzyme. 5 mM free valproic acid inhibited the activities of various acyl-CoA dehydrogenases only very weakly.

Derivation and Evaluation of an Equation for Prediction of Free Phenytoin Concentration in Patients Co-Medicated with Valproic Acid

The relationship between free phenytoin fraction (F-PHT-F) and valproic acid (VPA) was studied under two conditions: (a) in serum samples from 43 institutionalized patients (212 serial data points) at plateau steady state, and (b) in plasma samples from 50 outpatients coming for regular visits to our clinic throughout the day. Results for both groups led to identical linear regression equations relating F-PHT-F to VPA: F-PHT-F = 0.095 + 0.001 (VPA). Nevertheless, as expected, the resulting equation gave an unreliable prediction of F-PHT-F due to a variable physiological matrix. Substituting the parameter F-PHT-F by its equivalent (F-PHT/PHT) gave the equation F-PHT = [0.095 + 0.001 (VPA)] PHT. The predictive power of this equation was evaluated by the comparison of obtained and predicted F-PHT concentrations. For the combined patient group (n = 93), an excellent agreement (r = 0.972 and F = 999.9; p less than 0.001) was obtained. Although the empirically derived constants of the equation are not unique and may vary depending on the conditions of different methodologies, the fundamental relationship has been established and can be used to reliably predict F-PHT concentration from plasma VPA and PHT concentrations.

The effect of carbamazepine on valproic acid disposition in adult volunteers.

1. Pharmacokinetic parameters for valproic acid (VPA) were determined before and following 2 weeks of carbamazepine (CBZ) administration in five healthy male volunteers. Mean VPA dosage was 16.4 mg kg-1 day-1. CBZ dosage was started at 100 mg twice daily and increased after 1 week to a total daily dose of 300 mg. 2. After CBZ administration, mean VPA plasma clearance increased from 0.90 +/- 0.18 s.d. to 1.26 +/- 0.24 l h-1 (P less than 0.05) as did clearance of free VPA (20.8 +/- 7.6 to 37.0 +/- 13.6 l h-1). Mean VPA elimination rate constant increased from 0.051 +/- 0.011 to 0.067 +/- 0.011 h-1 (P less than 0.05) after CBZ administration. 3. Mean area under the serum concentration vs time curve decreased from 675.0 +/- 130.5 to 475.7 +/- 75.7 mg l-1 h (P less than 0.05) after CBZ administration. Mean serum VPA half-life decreased from 14.0 +/- 2.4 to 10.6 +/- 1.4 h (P less than 0.05). Mean serum VPA trough concentrations decreased from 44.0 +/- 16.7 to 27.0 +/- 10.4 micrograms ml-1 (P less than 0.05). 4. A significant change was not observed in the mean VPA volume of distribution after CBZ coadministration suggesting that enzyme induction rather than a competition for plasma protein binding sites was involved in this interaction. 5. Despite the increased clearance of VPA, the urinary recovery of VPA or conjugate did not increase after CBZ administration.

Pharmacokinetics of free and total sodium valproate in adolescents and young adults during maintenance therapy.

The pharmacokinetics of total and free valproic acid (VPA) in plasma and whole blood was investigated in seven adolescents and young adults (mean age 17.3 years) during a dosage interval at steady state. The concentration curves of VPA in whole blood after an oral morning dose (mean 8.2 mg/kg body wt.) closely followed those in plasma but at a reduced level. The apparent volume of distribution (Vd) of total VPA was 0.150-0.197 l/kg body wt. and of free VPA 0.911-1.58 l/kg body wt., which indicates considerable distribution of unbound VPA as well as drug binding to extravascular proteins. The terminal half-life of free VPA (6.4-6.7 h) was significantly shorter (P less than 0.05) than the half-life of total VPA (10.4-11.9 h). The binding of VPA in plasma was concentration dependent and fluctuated considerably within the individual dosage intervals. Concentrations of unbound VPA in plasma water of whole blood varied to a corresponding degree, since distribution to blood cells was low (mean 2.2%). It is concluded that there are substantial differences in the pharmacokinetics of free and total VPA. This may contribute to the well-known poor correlation between dose, plasma concentrations and effect of VPA.

Valproic acid developmental toxicity and pharmacokinetics in the rhesus monkey: an interspecies comparison.

This study was undertaken to assess the developmental toxicity and drug distributional and metabolic characteristics of prenatal valproic acid (VPA) exposure in rhesus monkeys. Oral administration of 20-600 mg/kg/day VPA (approximately 1-15 X human therapeutic dose) to 33 animals on variable gestational days (GD) during organogenesis resulted in dose-dependent developmental toxicity manifested as increased embryo/fetal mortality, intrauterine growth retardation, and craniofacial and skeletal defects. Biphasic plasma elimination curves were observed for total and free VPA on the first (GD 21) and last (GD 50) days of treatment in the 100- and 200-mg/kg/day dose groups. VPA exhibited dose-independent elimination kinetics at the plasma concentrations observed in this study. There was no significant change in pharmacokinetic parameters (maternal plasma elimination rate, area under the curve, peak plasma concentration) between the first and last days of treatment at either dose level. Placental transfer studies indicated that embryos were exposed to half the free VPA concentrations present in maternal plasma on GD 37. Comparisons of interspecies sensitivity to VPA-induced developmental toxicity in the mouse, rat, monkey, and man are made.

Variable free and total valproic acid concentrations in sole- and multi-drug therapy.

Total and free valproic acid (VPA) serum concentrations differ between patients on sole therapy (SOLE Group) and those taking multiple drugs (MULTI Group). We found significantly higher total and free VPA levels and free fractions in 25 SOLE patients than in 29 MULTI patients, both at morning predose (minimum) and postdose (maximum) testing. Results in SOLE versus MULTI patients were, respectively, as follows: total minimum 70.5 vs. 50.2 micrograms/ml (p less than 0.01); total maximum 106.8 vs. 89.5 micrograms/ml (p less than 0.05); free minimum 9.8 vs. 4.4 micrograms/ml (p less than 0.001); free maximum 18.9 vs. 12.0 micrograms/ml (p less than 0.01). The wide variation in total and free levels suggests the need to monitor VPA levels carefully. Not only does use of other drugs influence VPA kinetics, but the time since last dose also affects levels. Nonlinear binding causes large increases in free levels as total VPA concentration rises. We monitor free and total VPA at minimum and maximum along with evaluation of side effects and seizures for better interpretation of dosage requirements.

Serum protein binding of valproic acid in fetus-mother pairs throughout pregnancy: correlation with oxytocin administration and albumin and free fatty acid concentrations.

The protein binding (expressed as percent free drug fraction) of the antiepileptic drug valproic acid (VPA) was studied in 65 fetus-mother pairs from weeks 13 to week 41 of gestation. The fetal free fractions (expressed as percent of total concentrations) of VPA were exceedingly high (greater than 50%) during weeks 13 to 16 of gestation; these values decreased to 20% by week 20 and further decreased gradually to 10% at term. There was a highly significant negative correlation between free VPA fractions and fetal albumin concentrations. Maternal free fractions of VPA gradually increased from 10% during early pregnancy to 20% at term. The free fractions of VPA were significantly higher in mothers who had received oxytocin. Thus, protein binding in the fetus exceeded that of the mother at term, whereas the converse was true during early gestation. These results agree with previous in vivo findings. It is likely that the free concentrations in the mother determine the drug effects and toxicity in both the mother and the fetus. Intermittent drug administration--particularly of large single doses--could result in a transient increase of the free concentrations of VPA, particularly because of the strong concentration dependence of VPA protein binding. Increased free fractions can also be expected from increased concentrations of displacing agents of endogenous or exogenous origin (other drugs).

Determination of valproic acid in plasma or serum by solid-phase column extraction and gas-liquid chromatography.

A simple, fast, sensitive, and inexpensive method is described for routine therapeutic monitoring of both total and free valproic acid (VPA) at therapeutic concentrations in seizure patients. The method uses solid-phase (Bond Elut) columns for the preliminary extraction of 100 microliters plasma or serum and gas-liquid chromatography (GLC) analysis, using 10% Carbowax 6000 on a Chromosorb WAW 80/100 column and alpha-methyl-alpha-ethylcaproic acid as an internal standard. The extraction efficiency for VPA at a concentration of 70 micrograms/ml is 98 +/- 4%. The calibration curve is linear in the range of 1.0-280 microliters/ml. The detection limit of VPA is 1.0 micrograms/ml. Syva enzyme multiplied immunoassay technique (EMIT) Free-Level filters were used in the analysis of free VPA from plasma. The described Bond Elut X GLC (BE X GLC) method was used to determine total drug concentrations in 56 VPA-treated patients, and the results were compared with those obtained by conventional liquid-liquid extraction X GLC analysis. The 44 sera involved were also analyzed by EMIT. In addition, BE X GLC was compared with the Abbott TDx system on an additional 21 specimens. Statistical agreement was excellent between BE X GLC and TDx, but poor between BE X GLC and liquid-liquid extraction X GLC. Agreement with the EMIT method was intermediate.

Valproic acid disposition in rabbits during chronic treatment with Escherichia coli endotoxin.

The disposition of valproic acid (VPA) in rabbits was studied after chronic treatment with Escherichia coli endotoxin. Endotoxin (1-2 micrograms/kg) was administered daily to 10 male New Zealand white rabbits for 5 days. On day 5, 50 mg/kg of VPA was given iv during the time of the peak febrile response. Blood samples were drawn at appropriate time intervals and analyzed for free and total VPA levels as well as plasma proteins and free fatty acids. The data were compared with similar control experiments performed 2 weeks before and after endotoxin treatment. Pharmacokinetic analysis indicated that the changes in free VPA clearance after endotoxin were related to the change in the febrile response during chronic treatment (r = 0.77; p less than 0.05); that is, animals which developed tolerance to the febrile response showed elevated drug clearance, whereas nontolerant animals showed decreased clearance of VPA.

Determination of free valproic acid concentrations using the Amicon Micropartition MPS-1 Ultrafiltration System.

Free valproic acid concentrations were determined using the MPS-1 ultrafiltration device marketed by Amicon with one YMB membrane per device. Valproic acid binding to the membrane was 5.6% of the free valproic acid concentration. The free valproic acid concentration increased as serial ultrafiltrate volumes were collected, suggesting that only small ultrafiltrate volumes should be collected from a large initial sample. The protein content of the ultrafiltrate was acceptable and was within the tolerances stated by Amicon. There was no significant difference between free valproic acid concentrations determined within 3 h of sample collection and those determined after storage at 4 degrees C or -20 degrees C for 1 week. There was no significant difference between free valproic acid concentrations in serum and heparinized plasma samples collected simultaneously.