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PubMed2006

Method for lipoprotein(a) density profiling by BiEDTA differential density lipoprotein ultracentrifugation.

In this article, we demonstrate the analytical power of linking density gradient ultracentrifugation with affinity separations. Here we address some of the analytical challenges in the study of lipoprotein(a), (Lp(a)). The mean density distribution of Lp(a) was determined by a differential density lipoprotein profile (DDLP). For DDLP, the lipoprotein density distribution of a serum sample with elevated Lp(a) levels was determined by ultracentrifugation using a BiEDTA complex as a density gradient. Lp(a) was removed from a second aliquot of the same serum sample by carbohydrate affinity using wheat germ agglutinin (WGA). WGA was demonstrated to have high specificity for Lp(a) in a serum sample. This sample was ultracentrifuged to obtain a lipoprotein density distribution in the absence of Lp(a). A DDLP was obtained after subtracting the Lp(a)-depleted lipoprotein density profile from the untreated lipoprotein density profile. The DDLP methodology reported herein gives relevant information of the lipoproteins in serum such as density, isoform, and subclass characteristics. Lp(a) was quantitatively isolated from serum with a recovery efficiency of 82%. Lp(a) was purified by ultracentrifugation. Lp(a) retained its inherent density (1.086 g/mL) and immunoreactivity. The major outcome of this research was the effectiveness of using affinity separations coupled with density ultracentrifugation for the isolation of pure Lp(a) from serum and its isoform characterization based on density by DDLP.

Bismuth↗

PubMed1972

Isolation of human synovial-fluid hyaluronate by density-gradient ultracentrifugation and evaluation of its protein content.

1. A compound of hyaluronate and protein, called hyaluronate-protein was isolated from pooled human synovial fluids by caesium chloride density-gradient ultracentrifugation. 2. The isolated hyaluronate-protein was labelled with [(125)I]iodide and the following studies were done. (a) Ultracentrifugation in caesium chloride showed that the protein moiety ((125)I counts) and hyaluronate (hexuronate) sedimented together in the middle of the gradient. (b) The labelled hyaluronate-protein was treated with trypsin, and ultracentrifugation showed that peptide fragments ((125)I counts) were dispersed throughout the gradient, indicating proteolytic digestion. Hyaluronate sedimented in the middle of the gradient. (c) The labelled hyaluronate-protein was digested with streptococcal hyaluronidase, and ultracentrifugation showed that hyaluronate fragments were dispersed throughout the gradient, indicating digestion of the polysaccharide. The protein moiety, without attached hyaluronate, now sedimented at the top of the gradient. (d) Ultracentrifugation of labelled hyaluronate-protein in 4m-guanidinium chloride showed that protein and hyaluronate sedimented together. 3. These studies confirm that hyaluronate is combined with a small quantity of protein in normal human synovial fluid. A mild method for the rapid isolation of hyaluronate-protein in good yield is described.

Centrifugation, Density Gradient↗

PubMed1975

Structural and compositional changes attending the ultracentrifugation of very low density lipoproteins.

The effects of repetitive ultracentrifugation on the physical and chemical properties of very low density lipoproteins (VLDL) were investigated. VLDL recentrifuged one to seven times were characterized by chemical analyses, analytical ultracentrifugation and electron microscopy. The VLDL content of triglyceride was increased and the proportion of phospholipid decreased by ultracentrifugation. Recentrifugation of VLDL decreased the number of Sf-o 20-100 particles and generated particles of Sf-o greater than 400. The bulk of the material removed from VLDL by ultracentrifugation was lipoprotein having pre-beta mobility on paper electrophoresis, flotation rates of Sf-o 10-100 and a particle size of 300-400 A-O. Two ultracentrifugations separated an average of 14% of the starting VLDL protein. Characterization of the apoproteins in this material by polyacrylamide gel electrophoresis, gel chromatography, immunoprecipitation and amino acid analysis demonstrated a relatively high proportion of beta-apoprotein and relatively little C-apoproteins.

Adult↗

PubMed1981

Improved detection of viruses by electron microscopy after direct ultracentrifuge preparation of specimens.

We have adapted the Beckman Airfuge air turbine ultracentrifuge and the new EM-90 particle-counting rotor to improve detection by electron microscopy of viruses in clinical specimens. Samples were clarified by centrifugation, pelleted in the EM-90 rotor directly to Formvar-coated copper grids, and strained with 1.5% sodium phosphotungstate. Virus counts and endpoint titrations of serial dilutions of partially purified preparations of poliovirus, SA11 rotavirus, herpes simplex virus, and vaccinia virus showed an increase of ca. 1.5 log10 to 3.0 log10 over the virus titers of unconcentrated preparations of the same material. An increased yield of 14% more positive specimens for rotavirus was obtained after preparation of clinical samples by direct ultracentrifugation versus a method without virus concentration (82 versus 72). A prospective study showed that detection of adenoviruses, herpesviruses, and enteroviruses increased when specimens were prepared by direct ultracentrifugation. Direct ultracentrifugation with the EM-90 rotor in the Airfuge ultracentrifuge is a rapid concentration method which enhances the rate and yield of virus detection from clinical specimens by electron microscopy and is easily adaptable to a diagnostic virology laboratory.

Cytomegalovirus↗

PubMed1999

Ultracentrifugation and concentration of a large volume of serum for HCV RNA during treatment may predict sustained and relapse response in chronic HCV infection.

The ability to predict accurately a sustained response during therapy in patients with hepatitis C virus (HCV) infection is unavailable. The aim of this study was to differentiate, during therapy, patients who would relapse from those with a sustained response by ultracentrifugation for residual serum HCV RNA. Sixty-one specimens (from 32 patients) collected during interferon therapy were assessed by ultracentrifugation. All were negative using a quantitative polymerase chain reaction (PCR) (detection limit < or = 100 copies/ml). One-milliliter aliquots were ultracentrifuged at 23,000 x g (160 min), and then the nucleic acid pellet was extracted, precipitated, and resuspended. Qualitative PCR was carried out in quadruplicate using two separate 5'UTR primer sets (8 results/specimen). A specimen was positive if > or = 1 gels was positive compared to controls. At weeks 12 and 24, 9/9 (100%) sustained response patients were negative by ultracentrifugation. In the 23 relapse patients at week 12, 7/12 specimens were positive; at week 24, 7/14 were positive. Earlier time points could not differentiate the patients' eventual response to therapy. The predictive value of a positive ultracentrifugation test for relapse at week 12 or 24 was 100%. The predictive value of a negative test for sustained response was 62% and 50% at week 12 and 24, respectively. These preliminary results indicate that patients with an eventual sustained response will have no detectable serum HCV RNA by week 12 or week 24. A positive result is 100% predictive of relapse.

Adult↗

PubMed1971

Quantitation of serum lipoproteins by electrophoresis on agarose gel: standardization in lipoprotein concentration units (mg-100 ml) by comparison with analytical ultracentrifugation.

Lipoprotein electrophoresis on agarose gel has been modified to allow estimation of the absolute quantity of each fraction. The reproducibility of the method is illustrated by 12 determinations in a single day on serum from one normal subject: mean total dye uptake was 302 +/- 9 (sd "corrected dye units," and the percentages of beta-, pre-beta, and alpha-lipoprotein were 56.1 +/- 0.9, 29.1 +/- 0.4, and 14.8 +/- 0.7, respectively. Reproducibility over a period of 8 months was also demonstrated. Serum lipoproteins of five normal and 15 hyperlipidemic individuals determined by this technique were compared with values obtained by analytical ultracentrifugation. The correlation coefficients were: 0.993 for pre-beta-LP vs. VLDL, 0.978 for beta-LP vs. LDL, and 0.867 for alpha-LP vs. HDL. Lipoprotein values obtained by paper electrophoresis were also correlated with those of the analytical ultracentrifuge, but to a lesser degree (r = 0.956, 0.691, and 0.786, respectively). Values for LDL and VLDL which were measured by refractometry after preparative ultracentrifugation were very similar to those obtained from the analytical ultracentrifuge. Serum triglyceride concentration was highly correlated (r = 0.972) with the agarose values for pre-beta-LP; serum cholesterol concentration was correlated (r = 0.673) with beta-LP. It is proposed that the standard curves of the comparisons with the analytical ultracentrifugal values be used to convert the corrected dye units of electrophoresis on agarose gel to mg/100 ml of specific lipoprotein.

Adult↗

PubMed1993

[Evaluation and problems of ultracentrifugal technique for separation and analysis of serum lipoproteins: comparison with other analytical methods].

An ultracentrifugal technique for separating and analyzing serum lipoproteins was evaluated in comparison with analyses by electrophoresis using agarose-gel and polyacrylamide-gel. In general, the percent of pre beta- and beta-lipoproteins in electrophoresis was estimated higher than the percent of VLDL and LDL in ultracentrifugal method, while the percentage of alpha-lipoprotein in the former was estimated lower than that of HDL in the latter. In cases with abnormal lipoproteinemias, various discrepancies arose between the methods. For examples, pre beta- and beta-lipoproteins were estimated too high by the analyses with electrophoresis. The cholesterol content in HDL decreases in hypertriglyceridemia accompanied by an increase in triglyceride content. Therefore, when HDL cholesterol is determined by a polyanion method to assess the net HDL concentration in such cases, it is estimated to be low. Such errors are not only found in the determination of HDL cholesterol, but also in apoproteins in liver cirrhosis, because the composition of HDL apoprotein is markedly altered. Since the heterogeneity of lipoproteins separated by ultracentrifugation is characteristic in hereditary disorders of lipoproteins such as LCAT deficiency, the centrifugal technique is essential for lipoprotein analysis in such disorders. The disadvantages in ultracentrifugation are cross-contaminations among fractions, and removals of lipids and apoproteins from lipoprotein particles. Apo A-I and E proteins, and phospholipids were removed from the particles more rapidly than other components. From the results of repeated ultracentrifugation of HDL, 3% of apo A-I was estimated to be lost from the HDL during the centrifuge procedure.

Electrophoresis↗

PubMed1990

Metabolism of apoB-100 in lipoproteins separated by density gradient ultracentrifugation in normal and Watanabe heritable hyperlipidemic rabbits.

We have examined the capability of a previously developed compartmental model to explain the kinetics of radioiodinated apolipoprotein (apo) B-100 in very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL) separated by density gradient ultracentrifugation after intravenous injection of radioiodinated VLDL into New Zealand white (NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbits. Our model was developed primarily from kinetics in whole blood plasma of apoB-100 in particles with and without apoE after intravenous injection of large VLDL, total VLDL, IDL, and LDL. When the initial conditions for this model were assumed to be an intravenous injection of radiolabeled VLDL, the plasma VLDL and LDL simulations for NZW rabbits and the VLDL, IDL, and LDL simulations for WHHL rabbits were found to be inconsistent with the observed density gradient data. By adding a new pathway in the VLDL portion of the model for NZW rabbits and a new compartment in VLDL for WHHL rabbits, and by assuming some cross-contamination in the density gradient ultracentrifugal separations, it was possible to bring our model, which was based upon measurements of 125I-labeled apoB-100 in whole plasma, into conformity with the data obtained by density gradient ultracentrifugation. The relatively modest changes required in the model to fit the gradient ultracentrifugation data support the suitability of our approach to the kinetic analysis of the metabolism of apoB-100 in VLDL and its conversion to IDL and LDL based upon measurements of 125I-labeled apoB-100 in whole plasma after injection of radiolabeled VLDL, IDL, and LDL. Furthermore, the differences in kinetics observed by us between data from whole plasma and data from plasma submitted to ultracentrifugal separation from the same or similar animals highlight the fact that small variations that can occur in the separation of lipoprotein classes by buoyant density can lead to confusing results.

Animals↗

PubMed1964

STUDIES ON ISOLATED CELL COMPONENTS. XVI. THE DISTRIBUTION OF ACID PHENYL PHOSPHATASE ACTIVITIES IN RAT LIVER BREI FRACTIONATED IN THE ZONAL ULTRACENTRIFUGE.

The zonal ultracentrifuge has been used to separate the major components of rat liver brei (soluble phase, ribosomes, microsomes, mitochondria, membranous fragments, and nuclei) during one centrifugation, by using a 1200 ml sucrose gradient varying linearly with radius from 17 to 55 per cent (w/w) with a "cushion" of 66 per cent sucrose at the rotor edge at speeds up to 30,000 RPM. Liver brei was found to contain a family of phosphatases (phenol disodium phosphate substrate, sodium malonate buffers and Turgitol NPX, a non-ionic detergent). Activity maxima at pH 4.1 and 5.9 were observed in untreated brei prepared in 0.25 M sucrose. The addition of the non-ionic detergent Turgitol NPX selectively caused the release of considerable additional activity between these optima. The activity measured at pH 4.1 was primarily associated with the cytoplasmic granules, while the activities at pH 4.8, 5.4 and 5.9 were found in both soluble phase and particulate-mitochondria and membranous fractions. The activities present beyond the region of the gradient occupied by the soluble phase (sample layer) were all bound to particles sedimentable at 105,536 g (average) in the preparative ultracentrifuge. The data suggest that the different activities are not similarly distributed between soluble phase and particulate fractions. When the data are expressed in terms of specific activity, the area in the gradient between the microsomes and mitochondria now appears richest in all the acid phenyl phosphatase activities measured, while the soluble phase and larger particulate fractions appear relatively poor in activity. This part of the gradient is occupied by small, dense granules which may be the so called lysosomes. Pretreatment of the brei with Turgitol NPX prior to fractionation in the zonal ultracentrifuge resulted in the solubilization of acid phenyl phosphatase activities (almost all the activity was in the sample zone of the gradient) and the non-specific destruction of the formed elements of the brei. Essentially all of the activities present in the original brei measured under these conditions were recovered after zonal ultracentrifuge fractionations.

Acid Phosphatase↗

PubMed2004

Evaluation of the protein binding ratio of drugs by a micro-scale ultracentrifugation method.

Ultracentrifugation methods have been widely used for the determination of the free fraction of compounds in plasma, especially for lipophilic compounds. To estimate the effect of contaminated proteins in the "protein-free phase" fraction, 200 microL of human plasma was separated into three layers by ultracentrifugation at 436,000g for 140 min with a table-top ultracentrifuge. Twenty microliters of the middle layer was taken as the protein-free fraction. Major contaminated proteins were analyzed by liquid chromatography/electrospray ionization/tandem mass spectrometry (LC/ESI/MS/MS) and identified as albumin, alpha-1-antitrypsin, alpha-2-HS-glycoprotein, apolipoprotein E, and apolipoprotein A-1. alpha-1-acid glycoprotein was not detected. Contamination of albumin was 0.13% of that in plasma. Simulation analysis demonstrated that at an actual free fraction of 1% (protein binding ratio of 99%), the extent of overestimation of free fraction was just 13% and the apparent free fraction was 1.13%. Human plasma protein binding ratios of 10 drugs estimated by this method correlated well with reported values determined by other methods, such as ultrafiltration and equilibrium dialysis, with a correlation factor of 0.98 and a slope of 0.99. Collectively, our results indicate the reliability of this micro-scale ultracentrifugation technique for the evaluation of the protein binding of drugs despite a little contamination of albumin.

Adult↗

PubMed2001

A short-run new analytical ultracentrifugal micromethod for determining low-density lipoprotein sub-fractions using Schlieren refractometry.

We have developed a new analytical ultracentrifugal micromethod for the determination of serum low-density lipoprotein (LDL) subclasses directly from ultracentrifugal Schlieren scans. We have used special software for the analysis of this type of single-spin density-gradient ultracentrifugation. The flotation of LDL patterns was obtained by underlayering a physiological salt solution with serum or isolated lipoprotein fractions raised to a density of 1.3 g/mL in the spinning ultracentrifugation capillary band-forming cell. The repeated analysis of Schlieren curves of the same sample from 10 to 100 microL in the 60-100 min full-speed interval time resulted in quite reproducible results. We obtained quantitative results by measuring the Schlieren areas between the sample curves and the reference baseline curve by using computerised numerical and graphic techniques. The decomposition of the integrated curve was carried out using a nonlinear regression program followed by deconvolution algorithm analysis in order to determine the parameters of the composing Gaussian subclasses. The LDL particle concentrations were calculated from the area under the integral of the Gaussian curve using a calibration data constant. The flotation range of the LDL Schlieren curves in the cell was identified with serum from which LDL had been removed by means of precipitation reagents and with centrifugation of isolated LDL aliquots. With this technique, we measured the concentration of LDL and analysed its polydispersity without the need for preceding sequential isolation of the LDL. On the basis of the Schlieren curves, the LDL samples were either physically paucidisperse, having a symmetrical peak within a narrow density range, or were polydisperse, showing an asymmetrical pattern distributed over a broader density region. The described method proved to be useful for a clear and immediate visual presentation of the concentration values of the LDL and for the identification of the heterogeneity of LDL variants without the need for the preparative isolation of that density class.

Adult↗

PubMed1985

Failure of ultracentrifugation as a means of separating plasma free insulin from immunoglobulin fraction prior to radioimmunoassay.

It has been reported that ultracentrifugation of plasma will allow direct measurement of free insulin in the serum of diabetic subjects with insulin antibodies. To validate this method, we determined recovery of immunoreactive insulin and immunoglobulin G from the plasma of normal individuals after ultracentrifugation. The upper and middle fractions of plasma after ultracentrifugation were evaluated at several combinations of time and temperature (4 degrees C, 25 degrees C, or 37 degrees C for 3, 4 or 5 hours). None of these conditions effectively removed all immunoglobulin G without causing concomitant loss of insulin. We conclude that ultracentrifugation of plasma prior to radioimmunoassay cannot be used to reliably determine free insulin concentration in the plasma from subjects with circulating insulin antibodies.

Humans↗

PubMed2000

Analytical ultracentrifugation and the characterization of chromatin structure.

This mini review consists of two parts. The first part will provide a brief overview of the theoretical aspects involved in the two kinds of experiments that can be conducted with the analytical ultracentrifuge (sedimentation velocity and sedimentation equilibrium) as they pertain to the study of chromatin. In the following sections, I describe the analytical ultracentrifuge experiments which, in my opinion, have contributed the most to our understanding of chromatin. Few other biophysical techniques, with the exception of X-ray scattering and diffraction, have contributed as extensively as the analytical ultracentrifuge to the characterization of so many different aspects of chromatin structure. In the course of his scientific career, Professor Henryk Eisenberg has made many important contributions to the theoretical aspects underlying ultracentrifuge analysis, especially in the analysis of solutions of polyelectrolytes and biological macromolecules [H. Eisenberg, Biological macromolecules and polyelectrolytes in solution, Clarendon Press, Oxford, 1976]. As an example he has devoted some of his research effort to the characterization of chromatin in solution. This review includes these important contributions.

Animals↗

PubMed1996

New revolutions in the evolution of analytical ultracentrifugation.

The use of the biophysical technique of analytical ultracentrifugation has recently undergone a resurgence. The commercial availability of the Beckman optima XL-A and XL-I analytical ultracentrifuges along with the continued growth in computing ability and analysis software has led to the expanded use of analytical ultracentrifugation and its capabilities. The genetic revolution and the search for further understanding of macromolecular interactions have again brought analytical ultracentrifugation to the forefront of macromolecular characterization.

Macromolecular Substances↗

PubMed1986

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.

Blood Proteins↗

PubMed2004

Comparison of ultracentrifugation and nuclear magnetic resonance spectroscopy in the quantification of triglyceride-rich lipoproteins after an oral fat load.

BACKGROUND: The measurement of triglyceride (TG)-rich particles after an oral fat challenge has been used to provide a measure of risk for coronary artery disease independent of the fasting plasma triglyceride concentration. The analytical "gold standard" for measuring TG-rich lipoproteins uses density gradient ultracentrifugation; however, this technique is labor-intensive. Because of our need to perform numerous postprandial analyses of TG-rich lipoproteins for a large interventional study (Genetics of Lipid Lowering Drugs and Diet Network), we evaluated the use of nuclear magnetic resonance (NMR) spectroscopy for measuring TG-rich particles. METHODS: EDTA-blood samples were obtained 0, 3.5, 6, and 8 h after ingestion of an oral fat meal (89% of calories from fat) in 20 apparently healthy individuals. The plasma TG concentrations of chylomicron and chylomicron remnant/VLDL fractions were analyzed by ultracentrifugation and NMR spectroscopy. RESULTS: Comparison of all values (n = 78) by ultracentrifugation (x) and NMR (y) produced a linear regression equation of y = 0.979x - 0.035 mmol/L (R(2) = 0.90) for chylomicrons and y = 1.398x + 0.067 mmol/L (R(2) = 0.96) for the fraction containing chylomicron remnants and VLDL. Postprandial response of chylomicrons and chylomicron remnant/VLDL was similar, with maximum response occurring between 3.5 to 6 h regardless of method of measurement. CONCLUSION: Chylomicron and chylomicron remnant/VLDL fraction measurements obtained by NMR have a high degree of correlation with results produced by ultracentrifugation. NMR may therefore be suitable as an alternative method for the measurement of postprandial TG-rich lipoproteins in individuals consuming a high-fat meal.

Adult↗

PubMed1986

Apolipoprotein B determination in the dissolved precipitate obtained after precipitation of LDL with polyvinylsulphate. An alternative method for the determination of LDL apolipoprotein B without using ultracentrifugation.

Using a commercially available test for LDL cholesterol (Boehringer Mannheim), a method was developed for determination of LDL apolipoprotein B without using ultracentrifugation. The infranatant obtained by precipitation of serum with polyvinylsulphate was redissolved in a saline citrate solution and incubated with phospholipase A2, phospholipase C, phospholipase D or triglyceride lipase, respectively. When the saline-citrate redissolved precipitate was monitored by electron microscopy, it appeared as a fibriform network. The additional incubation with phospholipase A2, C, D, or triglyceride lipase resulted in a molecularization of LDL particles. By electron microscopy these particles could not be distinguished from LDL particles isolated by ultracentrifugation. In radial immunodiffusion tests, the additional incubation of the redissolved precipitate with phospholipase C or phospholipase D resulted in a total loss of the slight immunoreactivity observed before phospholipase incubation. However, additional incubation with triglyceride lipase resulted in a significant increase in immunoreactivity. Only the additional incubation of the redissolved precipitate with phospholipase A2 resulted in an immunoprecipitation reaction comparable to that with LDL particles isolated by ultracentrifugation. Using a resolubilized and phospholipase A2-incubated precipitate of a pool serum as apolipoprotein B standard, a good correlation was obtained between apolipoprotein B values measured in this dissolved precipitate and those measured in the d greater than 1.006 kg/l fraction isolated by ultracentrifugation (r = 0.95; y = 0.95x + 0.018; n = 44).

Apolipoproteins B↗

PubMed1984

The EMIT FreeLevel ultrafiltration technique compared with equilibrium dialysis and ultracentrifugation to determine protein binding of phenytoin.

A rapid new ultrafiltration technique (EMIT FreeLevel System I) for the routine measurement of unbound phenytoin concentrations was evaluated. The precision of this procedure was sufficient (between-days coefficient of variation, 11.7%). The results obtained by the ultrafiltration technique for the percentage of free phenytoin in samples from about 40 non-uraemic patients treated with this drug were in good agreement with those determined by ultracentrifugation and equilibrium dialysis (ultrafiltration vs ultracentrifugation, y = 0.94x + 0.60%; ultrafiltration vs equilibrium dialysis, y = 1.02x - 0.60%). The mean value of the results obtained by ultracentrifugation was significantly about 8% lower than that observed with equilibrium dialysis, apparently due to a sedimentation of free phenytoin during ultracentrifugation. With the methods used in our study, the mean values of percentage phenytoin bound to serum proteins obtained in samples from non-uraemic patients ranged from 91.8 to 92.8%. All 3 methods yielded similar binding curves for phenytoin, with spiked human pool sera containing albumin concentrations between 19 and 45 g/L. A rise of the unbound phenytoin fraction was observed with increasing total concentrations of the drug and a decrease of the albumin concentration. With samples from non-uraemic patients (n = 203), a rather good correlation was found between free and total phenytoin concentrations (r = 0.91). In a number of patients (n = 11), free phenytoin concentrations correlated better than total phenytoin concentrations with the clinical status. Patients with free phenytoin concentrations of less than or equal to 2.1 micrograms/ml and total phenytoin concentrations above the therapeutic range did not show signs of toxicity. From the results of our study it is concluded that the EMIT FreeLevel ultrafiltration technique is very well-suited for a rapid and reliable separation of unbound phenytoin. In patients with altered protein binding or an unusual clinical response, free phenytoin determinations appear to be necessary for proper interpretation of total phenytoin levels and rational dosage adjustment.

Adolescent↗