Fraction Of Phenytoin Research Articles

Protein binding of phenytoin and lidocaine in pediatric patients with type I diabetes mellitus.

We evaluated serum protein binding of phenytoin and lidocaine, the extent of albumin and hemoglobin glycation, and alpha-1-acid glycoprotein concentrations in 47 children and adolescents (9-17 years) with type I diabetes mellitus (DM). Serum was incubated with phenytoin (n = 47) and lidocaine (n = 32) to yield total concentrations of 19.4 +/- 1.34 and 4.6 +/- 0.5 mg/l, respectively. A serum ultrafiltrate was prepared from an aliquot of each sample by membrane centrifugation. Total and free concentrations of phenytoin (free fraction 8.3 +/- 1.0%) and lidocaine (free fraction 25.8 +/- 11.6%) were determined using a fluorescence polarization immunoassay technique. A linear relationship (r = 0.63, p = 0.0001, y = 40.1 + [-0.2x]) was also found between the alpha-1-acid glycoprotein concentration (68.5 +/- 20.9 mg%, range 47.2-134.7 mg%) and lidocaine-free fraction (n = 32). No relationship existed between the extent of glycated albumin and the lidocaine-free fraction, in contrast to the linear correlation (r = 0.49, p = 0.0005, y = 3.7 + 0.4x) found between the extent of glycated albumin and the free fraction of phenytoin in serum (n = 47). A similar correlation (r = 0.49, p = 0.014, y = 3.7 + 0.3x) was found between glycated albumin (%) and the phenytoin-free fraction (3.9 +/- 0.9%) when examined in a separate (n = 17) set of patient samples spiked to contain a total serum phenytoin concentration of 32.6 +/- 1.4 (range 29.4-35.7) mg/l. Our data demonstrate a predictable increase in the free fraction of phenytoin as the extent of albumin glycation increases in pediatric patients with type I DM who are in poor glycemic control. This relationship was not found for lidocaine, a representative basic compound. These findings suggest that glycation of albumin alters the binding of phenytoin in children and adolescents with type I DM.

Pharmacologic interactions between valproate and other drugs

Valproate is often administered with other antiepileptic drugs, a practice that can lead to clinically significant pharmacologic interactions. Concomitant administration of such enzyme-inducing anti-epileptic drugs as carbamazepine, phenobarbital, primidone, or phenytoin will markedly accelerate the metabolic conversion of valproate, particularly in children. In response to the effects of enzyme induction, valproate dosage may need to be doubled to maintain therapeutic serum levels. Valproate does not appear to induce enzymatic drug metabolism, but rather acts as a metabolic inhibitor. Because of this inhibition, phenobarbital dosage must often be reduced after valproate is added to the therapeutic regimen. Valproate also may markedly increase concentrations of the active epoxide metabolite of carbamazepine. The interaction between phenytoin and valproate results primarily from displacement from plasma proteins. The resulting increase in the free fraction of phenytoin alters the relationship between total phenytoin concentration and the drug's pharmacologic effect. Thus, clinical evidence of toxicity may be present at concentrations usually considered to be in the therapeutic range.

Dissolution and bioavailability of phenytoin in solid dispersion with phosphatidylcholine.

An attempt was made to improve the dissolution behavior of phenytoin (PHT), a poorly watersoluble drug, with phosphatidylcholine (PC) solid dispersion (SD). The powder X-ray diffraction patterns indicated that PHT was present in an amorphous state in SD when the molar fraction of PHT was under 0.33. Infrared spectra suggested a weak interaction, probably hydrogen bonding, between PC and PHT in SD. The solubility of PHT was 30μg/ml in both pH1.2 and 6.8 test solutions. The PHT concentration increased temporarily to 36μg/ml in both pH 1.2 and 6.8 test solutions with SD in which the molar fraction of PHT was 0.25 (SD (0.25)). This is only 1.2 times the PHT solubility, but the dissolution rate in pH 1.2 test solution was significantly improved: the PHT concentration reached 24μg/ml in the first 5min, whereas it was 6μg/ml in the case of PHT crystals. The dissolution rate was slightly higher even with physical mixture (PM) because of improved wettability.After oral administration of SD (0.25) to rabbits, plasma concentrations up to 8h were significantly higher than those in the cases of PM (0.25) and PHT crystals, but there was no significant difference in the area under the plasma concentration curve. PM (0.25) did not show improved bioavailability. These results were consistent with the results of the dissolution test at pH1.2. PM gave an increased plasma concentration of PHT if PC was used in a large excess over PHT (PM (0.10)), it was considered that PC functioned as an oil in this case.

Identifying patients who might benefit from free phenytoin monitoring.

Guidelines for measuring free drug concentrations in serum have become necessary due to the easy availability of these assays as a result of the introduction of commercial kits. The present study was performed to identify patients or groups of patients in whom the serum free phenytoin fraction varied from normal, such that they might benefit from measurement of serum free phenytoin. Three hundred fourteen samples submitted for routine phenytoin analysis were studied by enzyme-modified immunoassay technique (EMIT). Thirty-eight patients on phenytoin monotherapy and without other factors thought to affect protein binding of this drug had a mean (+/- SD) free phenytoin fraction of 9.8 +/- 1.8% of total concentration (mean serum albumin concentration 43.4 +/- 3.9 g/L). The free fraction was elevated by administration of comedications which are themselves highly protein bound, and in those patients who were hypoalbuminaemic (serum albumin less than 30 g/L). The groups studied were not mutually exclusive, but stepwise regression analysis showed that other factors known to affect serum albumin (e.g., age greater than 65 years, liver or renal disease, or pregnancy) did not, in themselves, produce a significant effect on free phenytoin fraction. Similarly, an elevated total serum phenytoin concentration was not a significant factor in producing an elevation in free phenytoin fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of phenytoin binding by glycosylation of albumin in IDDM.

We measured glycosylated albumin and hemoglobin and serum protein binding of phenytoin in 57 children and adolescents with insulin-dependent diabetes mellitus (IDDM). Serum was incubated with phenytoin to yield concentrations of 15 and 25 mg/L, and a serum ultrafiltrate was prepared from an aliquot of each sample. We observed a linear correlation between glycosylated albumin and the free fraction of phenytoin at serum phenytoin concentrations of 15 mg/L (r = .35, P = .03) and 25 mg/L (r = .40, P = .003). A better correlation existed between the free fraction of phenytoin and total albumin concentrations for both serum concentrations (r = .45, P = .005 for 15 mg/L; r = .56, P = 10-5) for 25 mg/L), whereas the best linear correlation resided between the free fraction of phenytoin and the concentration of nonglycosylated albumin (r = .54, P = .0005 for 15 mg/L; r = .63, P less than 10(-6) for 25 mg/L). There was no correlation between the free fraction of phenytoin and the concentration of glycosylated albumin. Incubation of solutions of glycosylated and nonglycosylated albumin demonstrated significantly lower binding to the glycosylated fraction (P = 8.1 X 10(-6)). These results indicate that glycosylation of albumin diminishes the affinity of the phenytoin binding site on albumin. This alteration may have clinical significance in that it may alter the disposition of phenytoin in patients with IDDM and produce free phenytoin serum concentrations that are not accurately reflected by total serum phenytoin concentrations.

The effects of valproate on plasma concentration of phenytoin in normal and D-galactosamine-treated rats.

The effect of valproate on the plasma concentration of phenytoin was examined in bothnormal and D-galactosamine-treated rats. In normal rats the total plasma concentration of phenytoin decreased and the plasma concentration of unbound phenytoin increased after the injection of valproate (300 mg/kg). By contrast, in rats showing low binding as a result of D-galactosamine treatment, the total and unbound concentrations of phenytoin in plasma were almost unchanged after the injection of valproate. Possible mechanisms to explain the above results are discussed in this paper on the basis of the results of plasma protein binding and tissue distribution studies. One such possible mechanism was thought to be that decrease in the total plasma concentration of phenytoin after valproate injection in normal rats may have resulted from an increased volume of distribution which is dependent on increase in the unbound fraction of phenytoin in plasma. On the other hand, one possible reason why valproate produced no significant influence on the total and unbound concentrations of phenytoin in plasma in the rats showing low binding may have been an unchanged volume of distribution of phenytoin preceding and following the injection of valproate which would have been dependent on the lack of change in the unbound fraction. The molar ratio of nonesterified fatty acid (NEFA) /albumin of the plasma protein was increased by D-galactosamine treatment. As NEFA is considered to be an important inhibitor of phenytoin binding to rat plasma, it could be speculated that valproate does not act as an inhibitor of phenytoin binding in rat plasma when the NEFA/albumin molar ratio of plasma protein is increased.

INTERACTION OF ISOFLURANE WITH THE BINDING OF DRUGS TO PROTEINS IN SERUM AND LIVER CELL CYTOSOL: An In Vitro Study

The influence of isoflurane on the binding to human serum and isolated human serum albumin (HSA), of drugs (diazepam, phenytoin and warfarin) highly bound to serum proteins, was studied in vitro by equilibrium dialysis. Distribution dialysis was used to determine the effect of isoflurane on the concomitant binding of the drugs to human serum and rat liver cytosol. Isoflurane (3.3% and 5%) significantly suppressed the binding of diazepam only to HSA by 70 and 81%, respectively. Under clinically relevant experimental conditions, isoflurane increased the free fractions of diazepam and phenytoin by 16 and 11%, respectively. During distribution dialysis 5% of the anaesthetic decreased the binding of both diazepam and phenytoin to human serum by 55%, whereas drug binding to liver cytosol was decreased by 36% for diazepam only. Isoflurane exercised no significant effect on the protein binding of warfarin. It is concluded that any interactions with drug protein binding during isoflurane anaesthesia are unlikely to be of clinical significance.

Micro-scale ultracentrifugation as an alternative to ultrafiltration for the determination of the unbound fraction of phenytoin in human serum.

Free phenytoin has been determined using micro-scale ultracentrifugation followed by analysis by EMIT. The effect of temperature on the determined free fraction was investigated and the ultracentrifugation procedure validated against ultrafiltration. Ultracentrifugation gave free fractions which were on average 16% lower than those obtained using ultrafiltration, but correlation was good, as was the correlation with measurements of total phenytoin (r = 0.90). Micro-scale ultracentrifugation is a simple procedure which can be of great utility in the measurement and investigation of free drug levels.

Effect of total drug concentration on the free fraction in uremic sera.

The effect of total drug concentration on the free fraction was studied in vitro using sera of uremic and nonuremic subjects. Both therapeutic and toxic concentrations of radiolabeled phenytoin, diazepam, and propranolol were used. The free fraction was separated by a pressure ultrafiltration method at 37 degrees C and determined from 200-microliters aliquots of serum and the ultrafiltrate. Sera from 11 acutely uremic and 10 chronically uremic patients and from 10 healthy control subjects were used. Only slight increases in the free fraction were found over the concentration range studied in both nonuremic and uremic subjects. The concentration-dependent increases in the unbound fraction of phenytoin and diazepam were quite negligible compared with the two- to threefold increases caused by uremia itself. Thus, variation in total concentrations of phenytoin, diazepam, and propranolol does not change the free fractions to a clinically significant degree even in uremic patients with a compromised binding capacity.

Postburn Serum Drug Binding and Serum Protein Concentrations

The free fractions of diazepam, imipramine, lidocaine, meperidine, phenytoin, propranolol, and salicylic acid were determined in the serum of seven burn patients (25% to 80% of their skin surface burned) about one week after the burn and in three of the patients at about four weeks following the injury. Serum protein fractions were measured by electrophoresis, and alpha-1 acid glycoprotein levels were determined. For the drugs that bind predominantly to albumin, the serum free fractions were greater in patients one week after the burn incident than in control subjects (diazepam, 0.055 vs. 0.017; phenytoin, 0.24 vs. 0.16; and salicylic acid, 0.69 vs. 0.32). The increase in free fraction for these drugs was attributed to the postburn decrease in serum albumin levels (2.2 vs. 4.4 g/dL, control). Imipramine, lidocaine, meperidine, and propranolol bind primarily to alpha-1 acid glycoprotein. The free fractions for these drugs decreased one week following the burn (imipramine, 0.074 vs. 0.095; lidocaine, 0.17 vs. 0.35; meperidine, 0.37 vs. 0.48; and propranolol, 0.045 vs. 0.107), presumably in response to the increased alpha-1 acid glycoprotein concentration (222 vs. 83 mg/dL, control). Diazepam, lidocaine, propranolol, and salicylic acid free fractions were still different from control values at four weeks after the accident.

Michaelis-Menten kinetics and the steady-state serum phenytoin/hydroxyphenytoin ratio.

The effect of nonlinear kinetics on the steady-state ratio of serum phenytoin (PHT) to total 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) was studied in epileptic patients. In each patient, the ratio increased in a nonlinear fashion in relation to both the PHT dose and the PHT serum level. Patients with longer apparent PHT half-lives (t50%) had a higher PHT/p-HPPH ratio. The t50% was either measured directly and found to correlate well with the patient's calculated Michaelis-Menten parameters (Vmax and Km), or was calculated from Vmax and Km values. A close linear correlation was found between the steady-state PHT/p-HPPH ratio and t50% values, interindividually and intraindividually, i.e., as the t50% increased with increasing PHT concentration in a given patient, the PHT/p-HPPH ratio increased in the same proportion. This indicates a good correlation between the overall Michaelis-Menten kinetics of PHT elimination and the PHT/p-HPPH ratio. A close linear correlation between a wide range of PHT doses and steady-state serum total p-HPPH levels was found, suggesting that when the PHT dose is lower than Vmax, the fraction of PHT undergoing parahydroxylation is constant. It is concluded that if, in a given patient, the p-HPPH level corresponds to the value expected from the PHT dose, it can be assumed that compliance and bioavailability are adequate and that the dose does not exceed Vmax. In that case, the PHT/p-HPPH ratio will provide a good estimate of the t50% of PHT at that particular PHT level.

Altered Drug–Serum Protein Binding in the Genetically Obese Zucker Rat

Drug–serum protein binding was evaluated in genetically obese Zucker rats, their lean littermates, and lean Sprague–Dawley rats. The free fraction (fp) of phenytoin was significantly higher in the obese rat (fp=0.177) compared to its lean littermate (fp=0.136), apparently due to displacement by free fatty acids. Conversely, diazepam and propranolol fp values were decreased in the obese Zucker rat (fp=0.107 and fp=0.122, respectively) compared to the lean Zucker rat (fp=0.140 and fp=0.174, respectively). Evidence strongly suggests that the increased binding of propranolol was not due to elevations in the serum concentrations of α1-acid glycoprotein (as is the case in the human obese population). Rather, the decreased fp for both diazepam and propranolol was a result of increased lipoprotein partitioning. Strain differences between the lean Zucker rat and lean Sprague–Dawley rat were also evident, with the serum binding of the Sprague–Dawley rat more closely resembling the obese Zucker rat.

Comparison of Equilibrium Dialysis, Ultrafiltration, and Gel Permeation Chromatography for the Determination of Free Fractions of Phenobarbital and Phenytoin

In this study, three techniques for measuring the free fractions of phenobarbital and phenytoin were compared: equilibrium dialysis, ultrafiltration, and the Hummel and Dreyer method for gel permeation chromatography. In their therapeutic range (15–40 and 10–20mg/L, respectively) the free fractions of phenobarbital and phenytoin were independent of the drug concentrations. Free fractions of phenobarbital as determined by equilibrium dialysis, ultrafiltration, and gel permeation chromatography were 58.7 ± 1.8, 58.3 ± 1.5, and 55.1 ± 1.7%, respectively. Free fractions of phenytoin were 18.1 ± 1.1, 17.0 ± 2.1, and 19.4 ± 1.2%, respectively. On lowering the albumin concentration, a similar increase in the free fractions of both drugs was observed with all three techniques. The results of this study show that all three techniques are suitable for the determination of free fractions of phenobarbital and phenytoin. Moreover, these techniques seem to be suitable for the investigation of physiological factors that may influence albumin drug binding.

Audit of a monitoring service for free phenytoin.

This study assessed the value of introducing the measurement of free phenytoin levels in a public hospital. After publicising the availability and purpose of the assay, free phenytoin levels were determined either (a) on the doctor's request or (b) when the total level was requested and the patient's record showed evidence of factors predisposing to an elevated unbound fraction. Total phenytoin was measured by EMIT, and the unbound fraction by ultrafiltration at 37 degrees C using [14C]-phenytoin as a tracer. During a 9 month period, 70 free level determinations were performed on 46 patients. These comprised 20% of all phenytoin assays. The median free phenytoin fraction was 13.6% (range 9.3-28.6%). While total phenytoin levels were below the normal optimum range in 61% cases, free levels were probably therapeutic or above in 70% cases. Dosage adjustments were recommended on the basis of the free level, and were followed more often when the doctor had requested the free level assay (P less than 0.05). The results suggest that a free phenytoin level assay can improve the usefulness of therapeutic drug monitoring, particularly when the doctor understands the purpose of the assay.

The variability of plasma protein binding of phenytoin in epileptic patients with hypoalbuminemia

The plasma protein binding of phenytoin in man is about 90% and the impaired plasma binding may lead to large variations in the unbound fraction of phenytoin in the plasma. In the present study, the percent of unbound phenytoin was accurately determined by equilibrium dialysis by using 14C-labeled phenytoin. When intra- and intersubject differences in the plasma binding were studied in 22 patients during long-term treatment with phenytoin, a strong linear correlation was observed between the unbound fraction of phenytoin, a strong linear correlation was observed between the unbound fraction of phenytoin and albumin concentration. The obtained results of the linear correlation were demonstrated to be of clinical significance in predicting the percentage of unbound phenytoin in patients with moderate hypoalbuminemia (plasma albumin of 2.0 to 3.0 g/100ml).

Monitoring free plasma concentrations of phenytoin.

British Journal of Clinical PharmacologyVolume 18, Issue 6 p. 971-973 Free Access Monitoring free plasma concentrations of phenytoin. GM Peterson, GM PetersonSearch for more papers by this authorS McLean, S McLeanSearch for more papers by this author GM Peterson, GM PetersonSearch for more papers by this authorS McLean, S McLeanSearch for more papers by this author First published: December 1984 https://doi.org/10.1111/j.1365-2125.1984.tb02576.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References Barth, N., Alvan, G., Borga, O. & Sjoqvist, F. (1976). Two-fold interindividual variation in plasma protein binding of phenytoin in patients with epilepsy. Clin. Pharmacokin., 1, 444– 452. DeMonaco, H. J. & Lawless, L. M. (1983). Variability of phenytoin protein binding in epileptic patients. Arch. Neurol., 40, 481– 483. Perucca, E. (1980). Plasma protein binding of phenytoin in health and disease: relevance to therapeutic drug monitoring. Ther. Drug Monit., 2, 331– 344. Peterson, G. M., McLean, S., Aldous, S., Von Witt, R. J. & Millingen, K. S. (1982). Plasma protein binding of phenytoin in 100 epileptic patients. Br. J. clin. Pharmac., 14, 298– 300. Rimmer, E. M., Buss, D. C., Routledge, P. A. & Richens, A. (1984). Should we routinely measure free plasma phenytoin concentration Br. J. clin. Pharmac., 17, 99– 102. Walther, H. & Meyer, F. P. (1979). Interindividual differences of protein binding in man. Int. J. clin. Pharmac. Biopharm., 17, 392– 395. Yacobi, A., Lampman, T. & Levy, G. (1977). Frequency distribution of free warfarin and free phenytoin fraction values in serum of healthy human adults. Clin. Pharmac. Ther., 21, 283– 286. Volume18, Issue6December 1984Pages 971-973 ReferencesRelatedInformation

Effect of valproate on free plasma phenytoin concentrations.

The plasma protein binding of phenytoin was studied in nine epileptic patients before and during addition of sodium valproate to the drug therapy. The free phenytoin fraction in plasma was significantly greater during sodium valproate treatment. The mean free fraction rose from 0.135 +/- 0.019 (s.d.) to 0.182 +/- 0.030. Total plasma phenytoin concentration fell significantly from a range of 4.3-26.2 micrograms/ml to 3.4-19.8 micrograms/ml during sodium valproate treatment. Neither the free plasma concentration nor the saliva concentration of phenytoin was significantly altered by sodium valproate. No significant correlation was found between plasma valproic acid concentrations and the change in phenytoin binding. We conclude that valproic acid displaces phenytoin from plasma protein binding sites but does not inhibit its metabolism.

Plasma concentrations of unbound phenytoin in the management of epilepsy.

In 46 epileptic patients the range of the unbound fraction of phenytoin in plasma measured by ultrafiltration (at 37 degrees C) and tracer-labelling with [14C]-phenytoin was 6.7%-33.3% with a median of 11.9%. The total and unbound phenytoin plasma concentrations were significantly correlated (r = 0.93, P less than 0.001), but in six patients the unbound concentration fell on or outside the 90% predictability limits for a single value. In all patients the unbound concentration reflected the clinical status of the patient equally or better than the total concentration. An inverse relationship was found between the plasma albumin concentration (within the normal reference range) and the phenytoin unbound fraction (r = -0.83, P less than 0.001) indicating that plasma albumin concentration is one of the important overall determinants of phenytoin protein binding. Saliva and plasma unbound phenytoin concentrations were significantly correlated (r = 0.98, P less than 0.001) but both collection of plasma samples and preparation of plasma ultrafiltrate using the Amicon micropartition system are simpler than collection and processing saliva, and interpretation of plasma unbound concentration does not require allowance for potential contamination. The additional value of the unbound phenytoin concentration in a clinically significant number of individuals would justify routine measurement of unbound phenytoin concentration in monitoring therapy, once available simplified methodology has been adequately characterised.

Should we routinely measure free plasma phenytoin concentration?

The plasma protein binding of phenytoin was investigated in 56 epileptic patients attending the outpatient clinic. The free phenytoin fraction was measured by equilibrium dialysis at 37 degrees C and the total concentration by a homogenous enzyme immunoassay technique. The free fraction ranged from 0.123 to 0.177 (median 0.144, mean +/- s.d. = 0.145 +/- 0.12). Distribution was consistent with normality. Four of the patients were also taking sodium valproate. The median free fraction of phenytoin in these patients was 0.174, 21% higher than that of the total group (P less than 0.05). The total concentration of phenytoin varied from 0.3 to 29.4 micrograms/ml (median 12 micrograms/ml, mean +/- s.d. = 13.31 +/- 6.13 micrograms/ml) and the free fraction was not related to the total drug concentration. There was a highly significant relationship between free phenytoin concentration and total phenytoin concentration (r = 0.986, P less than 0.001). There appears to be very little variability in protein binding of phenytoin in epileptic patients and thus total plasma phenytoin concentration closely reflects the free (unbound) drug concentration. Routine estimation of free plasma phenytoin concentration is therefore unnecessary and should be reserved for those patients where alteration in binding is likely, e.g. renal or hepatic disease or where adverse effects occur at unexpectedly low total phenytoin concentrations.

Monitoring phenytoin in salivary and plasma ultrafiltrates of pediatric patients.

The plasma binding of phenytoin and the relationship between phenytoin concentrations in salivary and plasma ultrafiltrates were evaluated in pediatric epileptic patients aged 2-15 years. Paired samples of plasma and saliva were ultrafiltered through an Amicon YMT membrane. Phenytoin concentrations were measured by a gas-liquid chromatography procedure. The phenytoin free fraction among pediatric epilepsy patients not taking valproic acid was normally distributed with a mean (+/- SD) of 9.2 +/- 1.8%. The mean (+/- SD) free phenytoin fraction among patients also taking valproic acid was 13.2 +/- 4.5%. Salivary ultrafiltrates exhibited a close correspondence with plasma ultrafiltrate concentrations, and the ratio of salivary to plasma ultrafiltrate concentrations was 1.06 +/- 0.15. Substantial intraindividual variation in the phenytoin free fraction and the increase in free fraction among patients on valproic acid emphasize the importance of using plasma unbound levels for monitoring phenytoin. The close agreement between plasma and salivary ultrafiltrate concentrations suggests that the latter will provide a practical noninvasive means of monitoring phenytoin.