Micropartition System Research Articles

Non-linear plasma protein binding of cannabidiol

BackgroundCannabidiol is highly bound to plasma proteins. Changes in its protein binding can lead to altered unbound plasma concentrations and result in alteration of pharmacological activity of cannabidiol-containing medications. This research has assessed non-linearity of cannabidiol plasma protein binding and the potential effect of tizoxanide on the binding.MethodCannabidiol protein binding was evaluated by ultrafiltration technique. Human plasma was spiked with cannabidiol stock solution to produce samples of various concentrations. For interaction study potential interactant tizoxanide was added in each sample. All samples were processed through Amicon Micropartition system and analyzed by HPLC.ResultsThe study has detected cannabidiol binding to borosilicate glass (9%) and polyethylene plastics (15%). In the interaction study the mean protein unbound fraction of cannabidiol was 0.05 (5%), indicating no binding interaction between cannabidiol and tizoxanide since cannabidiol unbound fraction without tizoxanide was also 5%. The cannabidiol fraction unbound was more than 2-fold greater at high concentrations compared to low concentrations.Conclusiona). At high concentrations cannabidiol plasma protein binding is non-linear. The non-linearity can affect elimination and medicinal effect of cannabidiol drugs. b). Borosilicate and polyethylene containers should be avoided in formulation, packing and administration of cannabidiol-containing medicines to guarantee correct doses. c). Cannabidiol medications can be co-administered with tizoxanide without caution.

Mode of Membrane Interaction and Fusogenic Properties of a de Novo Transmembrane Model Peptide Depend on the Length of the Hydrophobic Core

Model peptides composed of alanine and leucine residues are often used to mimic single helical transmembrane domains. Many studies have been carried out to determine how they interact with membranes. However, few studies have investigated their lipid-destabilizing effect. We designed three peptides designated KALRs containing a hydrophobic stretch of 14, 18, or 22 alanines/leucines surrounded by charged amino acids. Molecular modeling simulations in an implicit membrane model as well as attenuated total reflection-Fourier transform infrared analyses show that KALR is a good model of a transmembrane helix. However, tryptophan fluorescence and attenuated total reflection-Fourier transform infrared spectroscopy indicate that the extent of binding and insertion into lipids increases with the length of the peptide hydrophobic core. Although binding can be directly correlated to peptide hydrophobicity, we show that insertion of peptides into a membrane is determined by the length of the peptide hydrophobic core. Functional studies were performed by measuring the ability of peptides to induce lipid mixing and leakage of liposomes. The data reveal that whereas KALR14 does not destabilize liposomal membranes, KALR18 and KALR22 induce 40 and 50% of lipid-mixing, and 65 and 80% of leakage, respectively. These results indicate that a transmembrane model peptide can induce liposome fusion in vitro if it is long enough. The reasons for the link between length and fusogenicity are discussed in relation to studies of transmembrane domains of viral fusion proteins. We propose that fusogenicity depends not only on peptide insertion but also on the ability of peptides to destabilize the two leaflets of the liposome membrane.

Serum concentrations of calcium, phosphorus, magnesium and calciotropic hormones in donkeys

To provide reference values for serum biochemical variables that are used for evaluation of mineral metabolism in donkeys and compare values with those in horses. 18 donkeys and 18 horses. Total calcium (tCa), total magnesium (tMg), and inorganic phosphorus (P) concentrations were measured in serum samples via spectrophotometry. Ionized calcium (iCa) and magnesium (iMg) concentrations were quantified with selective electrodes. By use of a micropartition system, tCa and tMg were fractionated to separate protein-bound (pCa, pMg) and ultrafiltrable fractions. Complexed calcium (cCa) and magnesium (cMg) concentrations were calculated by substracting ionized fractions from ultrafiltrable fractions. Parathyroid hormone (PTH) and calcitriol (CTR) concentrations were measured via radioimmunoassay. Serum tCa concentration in donkeys (3.37 +/- 0.21 mmol/L) was composed of pCa (1.59 +/- 0.21 mmol/L [47.0 +/- 4.2%]), iCa (1.69 +/- 0.04 mmol/L [50.4 +/- 3.0%]), and cCa (0.09 +/- 0.08 mmol/L [2.6 +/- 2.9%]). Serum tMg concentration (1.00 +/- 0.08 mmol/L) was fractioned in pMg (0.23 +/- 0.08 mmol/L [23.4 +/- 8.1%]), iMg (0.59 +/- 0.04 mmol/L [58.8 +/- 5.1%]), and cMg (0.18 +/- 0.08 mmol/L [17.8 +/- 7.2%]). Serum concentrations of P (1.14 +/- 0.30 mmol/L), PTH (20.4 +/- 21.2 pg/mL), and CTR (13.4 +/- 5.9 pg/mL) were determined. Serum variables of mineral metabolism in donkeys were within reference ranges for horses. However, when compared with horses, donkeys had higher iCa, cMg, and CTR and lower pMg and PTH concentrations.

Fractionation of calcium and magnesium in equine serum

To establish reference values for protein-bound, ionized, and weak-acid complexed fractions of calcium and magnesium in equine serum and determine stability of ionized calcium (iCa) and ionized magnesium (iMg) in serum samples kept under various storage conditions. 28 clinically normal horses. Total calcium (tCa) and magnesium (tMg) in equine serum were fractionated by use of a micropartition system that allows separation of protein-bound calcium (pCa) and magnesium (pMg) and ultrafiltrable calcium (microCa) and magnesium (microMg) fractions. Serum concentrations of iCa and iMg were measured in the ultrafiltrate by use of selective electrodes. Serum concentration of complexed calcium (cCa) or magnesium (cMg) was calculated by subtracting iCa or iMg from microCa or microMg, respectively. Mean +/-SE serum tCa concentration was 3.26 +/- 0.06 mmol/L. Calcium fractions were as follows: pCa, 1.55 +/- 0.03 mmol/L (47.4 +/- 0.9%); iCa, 1.58 +/- 0.03 mmol/L (48.5 +/- 0.7%); and cCa, 0.13 +/- 0.02 mmol/L (4.1 +/- 0.9%). Serum tMg concentration was 0.99 +/- 0.04 mmol/L. Magnesium fractions were as follows: pMg, 0.33 +/- 0.04 mmol/L (33.3 +/- 4.2%); iMg, 0.57 +/- 0.02 mmol/L (57.6 +/- 1.7%); and cMg, 0.09 +/- 0.02 mmol/L (9.1 +/- 1.9%). Refrigeration (4 degrees C) did not affect iCa values, whereas iMg declined by 8% after 120 hours. Neither iCa nor iMg was affected by freezing (-20 degrees C). In equine serum, iMg is less stable than iCa; thus, when serum samples are not going to be analyzed promptly, freezing may be preferable to refrigeration for storage.

Fractionation of canine serum magnesium

Serum total magnesium (tMg) consists of 3 fractions: ionized magnesium (iMg), protein-bound magnesium (pbMg), and complexed magnesium (cMg). Serum iMg may be measured by an ion-selective electrode, but determination of pbMg and cMg has not been attempted in dogs. The objectives of this study were to assess the validity of a micropartition system to fractionate serum tMg and to establish reference intervals for pbMg, cMg, and iMg in clinically normal dogs using this method. Serum samples from 10 clinically healthy dogs were fractionated using a micropartition system (Centrifree YM-30, Amicon Corp, Lexington, MA, USA). Serum tMg and iMg were measured in whole serum, and tMg was also measured in the ultrafiltrate. Concentration of cMg was obtained by the subtraction of iMg from tMg concentrations of the ultrafiltrate. Protein-bound Mg was calculated by subtracting the tMg concentration of the ultrafiltrate from the tMg concentration of whole serum. Results for pbMg and cMg using the micropartition system showed good reproducibility. Determination of tMg and iMg had acceptable inter- and intra-assay precision. Concentrations of iMg, cMg, and pbMg were 0.50 +/- 0.05 mmol/L, 0.05 +/- 0.04 mmol/L, and 0.24 +/- 0.04 mmol/L, representing 63%, 6%, and 31% of the tMg concentration, respectively. The micropartition system was a reproducible means to accurately assess cMg and pbMg concentrations in dogs.

Development and validation of a method to determine the unbound paclitaxel fraction in human plasma

Paclitaxel is pharmaceutically formulated in a mixture of Cremophor EL and ethanol (1:1, v/v). The unbound fraction of the anticancer drug paclitaxel in plasma is dependent on both plasma protein binding and entrapment in Cremophor EL micelles. We have developed a simple and reproducible method for the quantification of the unbound paclitaxel fraction in human plasma. Human plasma was spiked with [ 3H]paclitaxel and [ 14C]glucose (unbound reference) and incubated at 37 °C for 30 min. Plasma ultrafiltrate was prepared by a micropartition system (MPS-1) and collected in a sample cup containing 100 μl of plasma to prevent the loss of paclitaxel due to adsorption. The radionuclides were separated after combustion of the biological samples using a sample oxidizer and the radioactivity was determined by liquid scintillation counting. The unbound fraction of paclitaxel was calculated by dividing the ratios of 3H and 14C in plasma ultrafiltrate and in plasma. The method was thoroughly validated using human plasma spiked with pharmacologically relevant concentrations of paclitaxel (10–1000 ng/ml) and Cremophor EL (0.25–2.0%). The method was precise, with a within-day precision ranging from 3.9 to 11.0% and a between-day precision ranging from 5.8 to 13.1%. In patient plasma with low serum albumin values containing 1% of Cremophor EL, the unbound fraction appeared to be significantly higher than that in plasma with normal albumin values. The determination of the unbound fraction of paclitaxel proved to be stable during a 10-week storage at −20 °C. Furthermore, the assay was applicable in patient samples. This assay can be used to determine the unbound fraction of paclitaxel in plasma. Moreover, its design should allow the determination of the unbound concentrations of other hydrophobic drugs.

Determination of calcium fractionation in dogs with chronic renal failure.

To determine concentrations of calcium (total [tCa], ionized [iCa], protein-bound [pCa], and complexed [cCa]) in dogs with chronic renal failure (CRF). 23 dogs with CRF. Serum calcium was fractionated by use of a micropartition system. Total calcium and iCa concentrations and pH were measured in unfractionated serum, and tCa concentration was measured in the ultrafiltrate. The pCa fraction was calculated by subtracting tCa of the ultrafiltrate from tCa concentration of unfractionated serum. The iCa concentration in unfractionated serum was subtracted from tCa concentration in the ultrafiltrate to determine the concentration of cCa. Concentrations of tCa, iCa, pCa, and cCa had wide ranges among dogs with CRF Dogs with significantly low tCa concentration (770 +/- 1.73 mg/dL) had cCa concentration (0.76 +/- 0.38 mg/dL) within reference range, whereas dogs with reference range to high tCa concentration (10.85 +/- 1.13 mg/dL) had significantly high cCa concentration (2.62 +/- 1.04 mg/dL). There was no significant difference in iCa or pCa concentrations between groups. Concentrations of tCa, iCa, cCa, and pCa varied widely in dogs with CRF Overall, cCa concentration was high, although subpopulations differed in cCa and tCa concentrations. Differences in tCa concentration were primarily attributable to differences in cCa fraction.

Valproic acid-ketoconazole interaction in normal, hypoalbuminemic, and uremic sera: lack of interaction in uremic serum caused by the presence of inhibitor.

Ketoconazole is an antifungal agent widely used in the management of patients with fungal infection, especially in patients with acute acquired immuno-deficiency syndrome (AIDS). Ketoconazole is 99% bound to serum albumin and may interact with valproic acid, an anticonvulsant with 90% to 95% binding to serum albumin. The interaction may be more significant in hypoalbuminemia, a common finding in patients with AIDS. However, valproic acid-ketoconazole interaction has not been reported. The authors prepared two serum pools from patients receiving valproic acid with normal serum albumin and another pool from patients with hypoalbuminemia. Another serum pool was prepared from uremic patients not receiving valproic acid. The aliquots of serum pool were supplemented with various concentrations of ketoconazole, representing therapeutic and slightly higher therapeutic concentrations. The concentrations of free valproic acid were determined in protein-free ultrafiltrates (prepared by centrifuging specimens at 25 degrees C with the Centrifree Micropartition System at 1500 g for 20 minutes) using fluorescence polarization immunoassay. In the serum pool with normal albumin concentration, the authors observed statistically significant displacement of valproic acid only at higher ketoconazole concentrations (10 and 20 micrograms/ml) whereas, in the serum pool with hypoalbuminemia, they observed statistically significant displacement of valproic acid by ketoconazole with lower and higher concentrations of ketoconazole. The magnitude of displacement was more significant at high valproic acid concentrations (95 and 150 mg/ml, respectively) probably because of the concentration-dependent binding of valproic acid to serum albumin. The authors observed no displacement of valproic acid by ketoconazole in the uremic serum pool. On the other hand, the free valproic acid concentrations were decreased in the presence of ketoconazole in the uremic serum pool.

A Study of Deproteinization Methods for Subsequent Serum Analysis with Capillary Electrophoresis

Fifteen different methods of serum protein removal are examined for applicability with subsequent serum analysis for low molecular mass metabolites and pharmaceutical agents by capillary electrophoresis (CE). Each of the deproteinization methods considered here has been reported in the literature and successfully used for serum protein removal in other applications. Two of the methods are found to be superior to the others for CE analysis. Protein removal by ultrafiltration with Centrifree Micropartition System filters and by precipitation using acetonitrile worked well for protein removal, as evidenced by reproducible (RSD <6%) determinations of several low molecular weight components. The former is capable of analyzing for free metabolites only, while the latter was found to be somewhat problematic with respect to evaporation of the acetonitrile, causing potential problems with reproducibility. Both techniques for protein removal were clearly suitable for the subsequent serum analysis by CE in untreated fused silica capillaries.

Serum protein binding of propofol in patients with renal failure or hepatic cirrhosis.

Serum protein binding is a limiting factor in the access of drugs to the central nervous system. Disease-induced modifications of the degree of binding may influence the effect of anesthetic drugs. The protein binding of propofol, an intravenous anaesthetic agent which is highly bound to serum albumin, has been investigated in serum samples from healthy volunteers, from patients with chronic renal failure not undergoing hemodialysis, from patients with chronic renal failure included in a regular hemodialysis program, and from patients with hepatic cirrhosis. Protein binding was determined by the ultrafiltration technique using an Amicon Micropartition System, MPS-1. The percentage of unbound propofol (mean(SD)) in healthy volunteers (n = 16) was 0.98 (0.48) % showing a high interindividual variability. Chronic renal failure did not significantly modify serum protein binding of propofol. In the chronic renal failure group not undergoing regular hemodialysis (n = 9), unbound propofol was 0.92 (0.34) %. In addition, patients in periodic dialysis did not show changes in propofol binding either compared before (1.11 (0.33) %; n = 13) or after hemodialysis (0.87 (0.38) %; n = 12). A slight decrease in albumin concentration was found in all renal patients (P < 0.05) in comparison to healthy volunteers. Creatinine and urea concentrations were higher in these patients (P < 0.01) but in the postdialysis group, the differences in urea levels were not significant when compared with those of volunteers. No changes in the degree of propofol binding were observed in patients with hepatic cirrhosis (0.97 (0.30) %; n = 14) when compared with the group of healthy volunteers. Significant differences were observed in albumin (P < 0.01) and bilirubin (P < 0.05) concentrations. Considering all subjects, the degree of binding did not correlate with biomedical data. Due to the absence of significant changes in the protein binding it is unlikely that there will be an exaggerated pharmacological response in patients with renal and hepatic disease following the administration of a standard propofol dose, although due to interpatient variability careful titration can be recommended.

Serum Ultrafiltrable Copper, Total Copper and Caeruloplasmin Concentrations in Gynaecological Carcinomas

Serum copper and caeruloplasmin concentrations are elevated in various carcinomas, but it is not known whether the rise in serum copper is due to elevation of caeruloplasmin alone or whether it is also due to the elevation of 'free' copper. We therefore determined ultrafiltrable copper to answer this question. Serum ultrafiltrate was prepared using the Amicon micropartition system and ultrafiltrable (free) copper was determined by flameless atomic absorption spectrophotometry. Serum total copper was determined by flame atomic absorption spectrometry and caeruloplasmin by rate nephelometry. There was no significant difference in the ultrafiltrable copper concentration between patients with gynaecological tumours and control subjects. However, total copper and caeruloplasmin concentrations were significantly elevated in cancer patients compared with control subjects. In contrast to total copper and caeruloplasmin concentrations, ultrafiltrable copper concentrations showed no significant increase with increase in the stage of the cancer.

Determination of N-Vinyl-2-Pyrrolidinone (NVP) in Rat and Dog Plasma by High-Performance Liquid Chromatography

Abstract A method for the determination of N-vinyl-2-pyrrolidinone (NVP) in both rat and dog plasma is described. The method involves the addition of an internal standard, a solution containing sodium lauryl sulfate, and an ultrafiltration step through a micropartition system which is subjected to centrifugation at 2000 × g for 15 minutes. The ultrafiltrate is then analyzed by an HPLC system with UV detection at 235 nm. The assay is linear in the concentration range of (0.2–8.0 μg/mL) for rat plasma with a minimum sensitivity of 100 ng/mL and a day to day variation of 3.0%. In the case of dog plasma, the assay is linear in the concentration range of (0.05–4.0 μg/mL) with a minimum sensitivity of 50 ng/mL when 100 μL aliquots of plasma are extracted. Application of this method to the protein binding studies of NVP to both rat and dog plasma is illustrated.

Determination of ultrafiltrable zinc in human milk by electrothermal atomic absorption spectrometry.

Percentages of non-protein-bound zinc in human milk have been reported by different workers, but ultrafiltration and zinc determination in human milk have not been comprehensively examined. However, zinc contamination and zinc membrane binding have been described for the determination of non-protein-bound zinc in serum. In this work, ultrafiltration was studied in terms of zinc contamination and zinc membrane binding. An MPS-1 micropartition system fitted with a YMT membrane was used. Zinc contamination was found to be less than 276 nmol dm-3 and the zinc recovery was 85 +/- 4%. The conditions for electrothermal atomic absorption spectrometry were also studied. The detection limit was found to be 26.4 nmol dm-3 and the upper linear range was 4 mumol dm-3. The precision varied from 3% (within-run) to 17% (between-run). The recovery of standard additions was 95 +/- 7% (n = 30, different human milk ultrafiltrate samples). Physiological values varied from 0.46 to 84 mumol dm-3 (4-56% of zinc in whole human milk). Expressed in mumol dm-3, zinc in human milk ultrafiltrate decreased slightly through the lactation period, whereas expressed as a percentage of the total zinc in milk, zinc in human milk ultrafiltrate remained constant from day 2 to day 69 post partum.

Comparison of Ultrafiltration Devices for Assessing Theophylline Protein Binding

The ability of two commercially available ultrafiltration devices to assess the protein binding of theophylline was compared using human serum albumin in pH 7.4 buffer at 37 degrees C. The devices compared were the Millipore-MC filter units (Catalog number UFC3LGC00) and the Amicon Centrifree Micropartition System; both rely on centrifugal force to separate the unbound and bound molecules. The results from each device were essentially identical provided that the Millipore units were centrifuged using conditions that would not allow heat buildup to occur and thereby cause a decrease in the binding. This problem could be minimized if the Millipore units were available in a low-binding 30,000 dalton cutoff membrane instead of the 10,000 dalton membrane currently available. The use of the Millipore 30,000 dalton cutoff polysulfone membrane (UFC3TTK00) reduced the centrifugation time necessary to obtain a sufficient volume of ultrafiltrate for assay, thereby minimizing the possibility of heat generation. Furthermore, the binding of theophylline to this device was low and comparable to the binding to the Centrifree devices. Because this low degree of membrane binding may not hold for other drugs, we would suggest that a Millipore unit supplied with a low-binding, 30,000 dalton cutoff membrane could provide binding data with an efficacy comparable to that achieved with the Centrifree devices.

Ultrafiltrable copper and related analytes in maternal and cord blood

We utilized the Amicon micropartition system (MPS-1) to prepare ultrafiltrates of serum for evaluating non-protein-bound copper (NPBCu). The method is rapid (a usual turnaround time less than 2 h), practical (only 1 mL of serum is required), and reproducible (average CV = 5.7% within assay and 9.3% between assays). Using this technique, we determined that (a) NPBCu in serum appeared to be fairly stable, showing little variation among subjects in different physiological conditions (normal female, pregnancy, or fetal samples); and (b) of all copper fractions studied, only NPBCu appeared to correlate between maternal concentrations and cord counterparts at delivery. Thus the NPBCu fraction may be appreciably involved in the overall mechanism of copper nutritional supply from mother to fetus.

An improved method for determining the nitrogen solubility of caseinates

SummaryThe nitrogen solubility index (NSI) method for the examination of caseinates was evaluated. It was found that widely scattering NSI values are mainly due to ineffective centrifugation and filtration of the caseinate dispersion. In order to avoid this, an improved combined centrifugation–filtration procedure was developed as an alternative to the original NSI method, using an Amicon micropartition system MPS-1 equipped with discs of filter paper. The micropartition devices can be centrifuged in a Gerber centrifuge of the type used for butyromctric fat determination. Several types of caseinates were evaluated with the new method and yielded a markedly smaller range of single values than were obtained with the NSI method.

Determination of Free Amino Acids in Wine by HPLC Using Precolumn Derivatization with Phenylisothiocyanate

An analytical method for the determination of free amino acids in wines is reported. It is based on their transformation into phenylthiocarbamyl derivatives. The derivatives were subjected to chromatography using an ODS column with a gradient elution program and ultraviolet detection. Prior to derivatization, two different clean-up methods of the sample were studied: the SEP-PAK C-18 system and a micropartition system. The method was applied to the study of variation of the amino acids during fermentation of sparkling wines.

Abstracts for the VI International Symposium on Human Purine and Pyrimidine Metabolism. Hakone, Japan, July 17-July 21, 1988.

The concentration of free and protein-bound urate in plasma was studied in control and gouty subjects. The uric acid bound to the plasma proteins was estimated by ultrafiltration method using Amicon's micropartition system. The total concentration of uric acid in the plasma and in the ultrafiltrate was analyzed by an enzymatic colorimetric method and the amount of uric acid binding was obtained from the difference between uric acid in plasma and in ultrafiltrate. In gouty patients the percentage of uric acid is quite the same that in control subjects, but it must be considered in relation to the higher values of uricemia; consequently, what is really increased is the free uric acid amount step by step with the increase of uricemia. So, the whole amount of free uric acid is available for the deposition in the tissue. The reduced binding capacity showed by gouty patients in comparison with healthy subjects is also confirmed by the results following to an hypoxanthine load per os, carried out in the two groups.

Pharmacokinetics of tiazofurin in dogs.

The pharmacokinetics of total and free tiazofurin, an antineoplastic agent, was studied in healthy mongrel dogs following intravenous and oral administration of the drug. The free fraction of tiazofurin was obtained from plasma by an ultrafiltration technique using a micropartition system. Total and free tiazofurin levels were determined by a sensitive high performance liquid chromatographic method. The percentage of tiazofurin bound to plasma proteins remained constant at approximately 15 per cent following administration to healthy mongrel dogs. The mean pharmacokinetic parameters of elimination rate constant (K), effective half-life (t 1/2), mean residence time (MRT) and the time to reach peak plasma level (tmax--after oral administration) were 0.32 +/- 0.04 h-1, 2.24 +/- 0.25 h, 3.23 +/- 0.36 h, and 1.78 +/- 0.50 h, respectively. The apparent volume of distribution at steady state was 0.98 +/- 0.30 1 kg-1 and the plasma clearance was 5.24 +/- 2.39 ml min-1 kg-1. About 90 per cent of tiazofurin was absorbed following oral administration. The pharmacokinetic parameters did not differ significantly between the total and free drug levels, indicating that the pharmacokinetics of total tiazofurin levels reflect those of the free tiazofurin in plasma.

Quantitation of macromolecular binding using size exclusion filters: Application to a fucosyltransferase assay

A method to quantify the covalent attachment of small radiolabeled substrates to macromolecules on the basis of molecular weight using size exclusion filters in an Amicon Centrifree micropartition system is described. GDP-[ 14C]- l-fucose was covalently attached to asialofetuin in a fucosyltranferase reaction catalyzed by mouse spermatogenic cell extracts. Radiolabeled product was separated from unreacted substrate by centrifuging 200–400 μl of cell extract through a 10-kDa size exclusion filter at 1000 g for 10 to 20 min. After 10 washes with an appropriate buffer, no detectable radioactivity was found in the eluant and the membranebound radiolabeled product was counted in a scintillation vial. Using this method the fucosyltransferase activity of mouse spermatogenic cells was approximately 17 pmol/mg protein/min which is essentially identical to values obtained using size exclusion chromatography. This technique provides a rapid, efficient, and inexpensive alternative for the isolation and detection of acceptor-substrate complexes.