Micro-Scale Ultracentrifugation as an Alternative to Ultrafiltration for the Determination of the Unbound Fraction of Phenytoin in Human Serum

1986 ◽  
Vol 23 (5) ◽  
pp. 603-607 ◽  
Author(s):  
L Jack ◽  
C Cunningham ◽  
I D Watson ◽  
M J Stewart
Keyword(s):  
Human Serum ◽  
Simple Procedure ◽  
Free Fraction ◽  
Free Drug ◽  
Effect Of Temperature ◽  
Unbound Fraction ◽  
Drug Levels ◽  
Micro Scale ◽  
Great Utility

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.

scholarly journals Comparison of Microscale Ultracentrifugation and Equilibrium Dialysis for the Determination of the Unbound Fraction of Theophylline in Human Serum

1989 ◽  
Vol 26 (2) ◽  
pp. 185-188 ◽  
Author(s):  
K McKenzie ◽  
I D Watson ◽  
M J Stewart
Keyword(s):  
Human Serum ◽  
Equilibrium Dialysis ◽  
Cost Effective ◽  
Free Drug ◽  
Effective Technique ◽  
Unbound Fraction ◽  
Cost Effective Technique ◽  
Dialysis Method

A microscale ultracentrifugation procedure for the measurement of free theophylline in serum is described and evaluated by comparison with an equilibrium dialysis method. Provided an ultracentrifuge is available, micro-ultracentrifugation is a simple, cost-effective technique for the routine determination of free drug fractions.

Determination of drug levels in human serum by circular dichroism

Chirality ◽  
1998 ◽  
Vol 10 (6) ◽  
pp. 507-512 ◽  
Author(s):  
Pilar Gort�zar ◽  
Alfredo Ro�n ◽  
Jes�s T. V�zquez
Keyword(s):  
Circular Dichroism ◽  
Human Serum ◽  
Drug Levels ◽  
Circular Dichroïsm

scholarly journals Influence of Assay Methodology on the Measurement of Free Serum Ceftriaxone Concentrations

1998 ◽  
Vol 42 (9) ◽  
pp. 2259-2261 ◽  
Author(s):  
Sue J. Kohlhepp ◽  
David N. Gilbert ◽  
James E. Leggett
Keyword(s):  
Human Serum ◽  
Binding Sites ◽  
High Performance ◽  
Species Differences ◽  
Free Fraction ◽  
Assay Method ◽  
Plot Analysis ◽  
Free Serum ◽  
Free Plasma

ABSTRACT The influence of assay methodology on the measurement of the active free fraction of ceftriaxone in plasma was determined. The free fraction was measured by three methods: agar diffusion bioassay, precipitation of plasma protein with methanol followed by high-performance liquid chromatography (HPLC) of the supernatant, and ultrafiltration of plasma followed by HPLC of the filtrate. In human serum, the free ceftriaxone levels were significantly lower (P = 0.03) when measured on ultrafiltrates compared to the other two methods. This difference disappeared when dolphin serum was studied. After ultrafiltration, human serum was shown, by Scatchard plot analysis, to have two ceftriaxone binding sites. Species differences were also demonstrated. Hence, in humans, determination of free plasma ceftriaxone varies with the assay method employed.

The Worthington "ultrafree" device evaluated for determination of ultrafiltrable calcium in serum.

1981 ◽  
Vol 27 (3) ◽  
pp. 466-468 ◽  
Author(s):  
J Toffaletti ◽  
D Tompkins ◽  
G Hoff
Keyword(s):  
Human Serum ◽  
Room Temperature ◽  
Reference Intervals ◽  
Effect Of Temperature ◽  
Healthy Population ◽  
Calcium Citrate ◽  
Coefficients Of Variation ◽  
Disposable Device

Abstract We evaluated a commercially-available disposable device ("Ultrafree," Worthington Diagnostics) for the anaerobic preparation of protein-free ultrafiltrates from serum for measurement of ultrafiltrable calcium. Sufficient filtrate for the analysis is obtained with 10 min from 0.2 to 0.4 mL of serum at room temperature. We assessed these ultrafilters with regard to permeability of calcium citrate, exclusion of proteins, frequency of leakage, and effect of temperature on results. Within-run and day-to-day coefficients of variation for human serum pools were 1.2 and 1.5%, respectively. Reference intervals (in mmol/L) for total (2.16-2.58), ultrafiltrable (1.44-1.67), dialyzable (1.25-1.41), and ionized (1.04-1.25) calcium have been determined for a healthy population of 69 women and 81 men, ages to 18 to 65 years. The device appears to be the most practicable yet available for use in making this measurement.

scholarly journals Characterization of discrete classes of binding sites of human serum albumin by application of thermodynamic principles

1994 ◽  
Vol 302 (1) ◽  
pp. 69-72 ◽  
Author(s):  
S Urien ◽  
P Nguyen ◽  
S Berlioz ◽  
F Brée ◽  
F Vacherot ◽  
...  
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Hydrophobic Interactions ◽  
Free Ligand ◽  
Effect Of Temperature ◽  
Binding Data ◽  
Different Temperatures ◽  
Acidic Ligands

The binding interactions of four ligands differing in acid-base properties with human serum albumin (HSA) were examined as a function of temperature. Binding to HSA decreased with increasing temperature for all four ligands. The bound and free ligand concentrations obtained at different temperatures were satisfactorily fitted to a model that incorporates the effect of temperature as an independent covariable and that directly allows the estimation of the enthalpic and entropic components of the ligand-albumin interaction, along with the precision of this estimation. Using this analysis, the binding of acidic ligands could be resolved into two classes of saturable sites, with the determination of the corresponding number of sites, whereas interpretation of binding data at each isolated temperature allowed only the determination of one saturable plus one non-saturable class of site. The thermodynamic constants indicate that binding of ionizable ligands to HSA involves electrostatic plus hydrophobic interactions, whereas only hydrophobic interactions are involved in binding to a second low-affinity class of site when present. Binding of non-ionizable ligands resembles that of the second class of low-affinity sites of ionizable ligands.

scholarly journals Microscale ultrafiltration technique for determining free drug in 50-microL serum samples.

1984 ◽  
Vol 30 (6) ◽  
pp. 878-879 ◽  
Author(s):  
W Wittfoht ◽  
K Duwe ◽  
W Kuhnz ◽  
H Nau
Keyword(s):  
Valproic Acid ◽  
Free Fraction ◽  
Free Drug ◽  
Laboratory Animals ◽  
Small Laboratory ◽  
Serum Samples

Abstract This ultrafiltration technique allows determination of free drug in 50 microL of serum. We ultrafiltered sera containing the following drugs--valproic acid (and its major metabolites), phenobarbital, diazepam, indomethacin, phenytoin, furosemide, and chloramphenicol--using both the commercially available micropartition system (MPS-1, Amicon), which requires a 200-microL sample, and our modified micro system, which requires only 50 microL. The value for the free fraction of each drug obtained in the two experiments agreed well. The smaller sample requirement makes the micro method particularly suited for pediatric samples and studies on small laboratory animals.

The Effect of Sample Handling on Free Valproic Acid Levels

2020 ◽  
Author(s):  
Dan Wang ◽  
Elizabeth Champion-Lyons ◽  
Ron Neyens ◽  
Nicole Bohm ◽  
Brittany Caddell ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Anticonvulsant Drug ◽  
Free Fraction ◽  
Free Drug ◽  
Reference Laboratory ◽  
Negative Bias ◽  
Sample Handling ◽  
Drug Levels ◽  
Parallel Testing ◽  
Significant Bias

Abstract Background Valproic acid (VPA) is a broad-spectrum anticonvulsant drug. Under normal conditions, this drug is highly protein bound. However, in patients with hypoalbuminemia, the free fraction can increase substantially while the total VPA levels remain in therapeutic range. The neurologic activity and toxicity of the drug are directly related to free drug levels. Methods Our in-house free VPA assay was validated using 20 patient samples obtained from a reference laboratory (RL1). It was further evaluated by parallel testing with RL1 using samples collected from our patients. Subsequently, sample handling effects were investigated by comparing free VPA levels measured in our laboratory to 3 selected RLs with different sample transportation conditions. Results No significant bias was observed between the in-house assay (y) and RL1 (x) assay in free VPA measurement (y = 1.12x + 0.072, r = 0.994). However, patient samples collected in our institution and sent to RL1 revealed significant negative bias (y = 0.776x − 3.861, r = 0.954). A large discrepancy in free VPA levels was further observed from identical aliquots of the same samples transported to 3 RLs in different conditions. Conclusions Our study demonstrated that sample handling has significant impact on free VPA levels. The observed magnitude of variation exceeds a clinically acceptable limit and could alter clinical decisions.

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