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Release of interleukin-8 and calprotectin during and after paediatric cardiopulmonary bypass with and without ultrafiltration.

Release of calprotectin and interleukin-8 (IL-8), changes in leukocyte counts and subsets and influence of extracorporeal ultrafiltration were evaluated during and after cardiopulmonary bypass (CPB) in 18 children undergoing open-heart surgery for congenital heart anomalies. Ultrafiltration was used in nine cases and nine were controls. Calprotectin concentration rose after start of CPB, peaking 48 hours postoperatively, with no significant intergroup difference. Positive correlation was found between duration of CPB and calprotectin (peak level and accumulated total). Circulating IL-8 was detected in all patients perioperatively, peaking at wound closure in the ultrafiltration group and at termination of bypass in the controls. CPB duration correlated significantly to peak level and accumulated total of IL-8. Seven of nine ultrafiltrate samples contained IL-8 at levels similar to the plasma concentration. Changes in white cell counts were mainly attributable to neutrophils. The two subgroups did not differ significantly in neutrophil counts. Neutropenia found after 10 minutes of CPB was replaced by neutrophilia, with maximal values postoperatively. Calprotectin and IL-8 thus were released into the circulation during CPB in children. Ultrafiltration did not affect the plasma concentrations of these substances, and only IL-8 was detected in the ultrafiltrate.

Calcium-Binding Proteins↗

Ultrafiltration of the waste plasma effluent from cardiopulmonary bypass circuit contents processed with a cell-washing device.

Blood conservation methods are commonly practiced throughout most hospitals that conduct cardiothoracic surgery. In an effort to reduce patients' exposure to homologous blood products and due to cost effectiveness of blood conservation techniques, this present study combines autotransfusion of the remaining blood in the extracorporeal circuit and ultrafiltration of the plasma effluent, and describes the resulting product. Seven patients, greater than 19 years of age, requiring cardiopulmonary bypass (CPB) were incorporated into this study. Exclusion criteria included age limitation. At termination of CPB, the remaining blood in the circuit was transferred to an autotransfusion machine and processed. Plasma (1054 +/- 206 ml) effluent was collected directly from the centrifugal bowl and processed through a ultrafiltrator, with a constant flow rate and negative pressure, until the plasma effluent concentrated down to an end processed volume of approximately 150 ml. The following variables were either measured or calculated: plasma-concentrate volumes per three minute interval, inlet/outlet pressures of an ultrafiltrator, transmembrane pressure (TMP), plasma free hemoglobin, fibrinogen, total protein, and colloid osmotic pressure. The average ultrafiltrate volume taken off from the plasma effluent was 828 +/- 237 ml, with an average ultrafiltrate volume of 115 ml in every three minute interval. The TMP did not change over the first 15 minutes of processing but became significantly elevated at the 18th minute interval and continued to increase and reach a maximum TMP of 286.5 +/- 2.1 mmHg at the end of concentration. Fibrinogen levels increased from pre-concentration values of 118.2 +/- 64 to 317 +/- 177 mg/dl (p = .03) along with increases in plasma free hemoglobin from 97.7 +/- 46 to 402.1 +/- 180 mg/dl (p = .0002). The total protein concentration increased by over 330% from baseline values. Ultrafiltrating plasma effluent from autotransfused cell salvaged CPB circuit contents could prove beneficial, but further study is required to discover ways to separate unfavorable products, such as activated platelet-leukocyte products and reduced plasma free hemoglobin, and to lower heparin concentrations of the plasma-concentrate.

Blood Transfusion, Autologous↗

The relationship between ultrafiltrate volume with icodextrin and peritoneal transport pattern according to the peritoneal equilibration test.

OBJECTIVE: To establish a relationship between peritoneal transport membrane pattern, analyzed by the peritoneal equilibration test (PET), and drained volume using icodextrin (7.5% Ico) and glucose (3.86% Glu) solutions. DESIGN: Thirty peritoneal dialysis patients were submitted to a standard 4-hour PET and divided into 4 transport categories based on dialysate-to-plasma ratio of creatinine (D/Pcr) and dialysate ratio of glucose at 4 and zero hours of the dwell (D4/D0). Patients were asked to perform exchanges for 2 consecutive nights in 10-hour dwells (2 L 3.86% Glu solution on the first night, and 2 L 7.5% Ico solution on the second night). The drained volume was measured and dialysate samples from the overnight exchanges were obtained for beta2-microglobulin (B2M) levels. RESULTS: PET classification using D/Pcr showed that 46.6% of the patients were high and high-average transporters, or 23.3% when D4/D0 was used. In spite of this difference, both methods showed significant correlation (p = 0.0001, r = 0.862). The mean drained volumes were similar for both solutions (for 3.86% Glu, 2696 +/- 369 mL; for 7.5% Ico, 2654 +/- 424 mL). The high and high-average transport patients classified by D4/D0 achieved a higher ultrafiltration with 7.5% Ico than with 3.86% Glu (p = 0.0235). When classified by D/Pcr, the difference was not significant (p = 0.2243). In the low and low-average transport patients classified by D/Pcr, we observed a significantly lower ultrafiltration when 7.5% Ico was used compared to 3.86% Glu solution (p = 0.0197). Using D4/D0, we saw a tendency toward lower ultrafiltration (p = 0.0719) in the same group. We then correlated the PET results and the difference between drained volume with 7.5% Ico and 3.86% Glu solution [deltaV (I-G)]. We found a significant negative correlation between D4/D0 and deltaV (I-G) (p = 0.002, r = -0.5390), and a positive correlation between D/Pcr and deltaV (I-G) (p = 0.005, r = 0.4932). The levels of B2M obtained with 7.5% Ico were higher than those obtained with 3.86% Glu solution (for 7.5% Ico, 9.47 +/- 6.71 microg/vol; for 3.86% Glu, 7.29 +/- 4.91 microg/vol; p = 0.004). Furthermore, we found significant correlation between the total amount of B2M obtained with 7.5% Ico solution and D4/D0 (p < 0.0001, r = -0.4493), and D/Pcr (p < 0.0001, r = 0.5431). CONCLUSION: Mean drained volume was similar between the two solution groups. High transporters, as defined by D4/D0, achieved higher ultrafiltration with 7.5% Ico than with 3.86% Glu solution. This is most likely due to the higher number of small pores in the peritoneal membrane. Low transporters, as classified by D/Pcr, achieved lower ultrafiltration with 7.5% Ico than with 3.86% Glu solution. The deltaV (I-G) and the PET results showed significant correlation, confirming that high transporters have a higher ultrafiltration volume with 7.5% Ico. The total B2M mass obtained with 7.5% Ico was greater than with 3.86% Glu solution and significantly higher in the high transport patients, indicating a larger number of small pores. Thus, the deltaV (I-G) could give us an idea of the peritoneal transport pattern in peritoneal dialysis patients.

Adult↗

The roles of complement factor C5a and CINC-1 in glucose transport, ultrafiltration, and neutrophil recruitment during peritoneal dialysis.

BACKGROUND: In a recent experimental study, we showed that low molecular weight heparin improved ultrafiltration and blocked complement activation and coagulation in a single peritoneal dialysis (PD) dwell. OBJECTIVE: The aim of the present study was to evaluate the possible contribution of the complement factor C5a and the potential interactions between C5a, the coagulation system, and cytokines of the interleukin (IL)-8 family (cytokine-induced neutrophil chemoattractant; CINC-1). METHODS: Nonuremic rats were exposed through an indwelling catheter to a single dose of 20 mL glucose-(2.5%) based filter-sterilized PD fluid, with or without the addition of anti-rat C5 antibody. The dwell fluid was analyzed 2 and 4 hours later concerning activation of the coagulation cascades, neutrophil recruitment, ultrafiltration volume; CINC-1, glucose, urea, and histamine concentrations; and ex vivo intraperitoneal chemotactic activity. RESULTS: The numbers of neutrophils and levels of thrombin-antithrombin complex (TAT) and CINC-1 increased significantly during the PD dwell. C5 blockade significantly reduced the levels of TAT and increased the ultrafiltration volumes at 2 hours. Glucose concentrations were significantly positively correlated to ultrafiltration volumes. CONCLUSIONS: Blockade of C5 leads to an increase in ultrafiltration, probably by a mechanism that involves a reduction in glucose transport. This effect may form a basis for improving PD efficiency in situations where high glucose transport limits ultrafiltration. Mechanisms connected to complement activation during PD may involve coagulation. Further studies of the intraperitoneal cascade systems under conditions of PD are indicated.

Animals↗

Protein binding of sotalol enantiomers in young and elderly human and rat serum using ultrafiltration.

The protein binding of sotalol (STL) enantiomers was evaluated using an ultrafiltration technique with serum from young (32 +/- 2 years, n = 5) and elderly (73 +/- 6 years, n = 5) male and female humans, and young (8 weeks, n = 4) and elderly (60 weeks, n = 3) male Sprague-Dawley rats. Serum samples were collected and immediately frozen at -20 degrees C. Within 1 week, the serum samples were thawed at room temperature, and adjusted to pH 7.4 using 0.05 M phosphate buffer, pH 5.0. Aliquots were spiked with 250 ng mL-1 and 500 ng mL-1 of each STL enantiomer, placed in ultrafiltration sets (Microsep, 30K molecular weight cut-off), capped, equilibrated to 37 degrees C, and centrifuged at 1850g for 1.5 h at 37 degrees C. Aliquots of ultrafiltrate and unspun serum were analysed for STL enantiomer concentration using a stereospecific HPLC assay. In all groups, bound fraction was less than 7% for both STL enantiomers. There were no significant differences in bound fraction between groups, or between enantiomers. Adsorption of STL enantiomers to the ultrafiltration device and membrane, evaporative loss of serum samples during centrifugation, and protein concentration in each ultrafiltrate sample were all negligible. It is concluded that the binding of STL in human and rat serum at therapeutic concentrations and physiological temperature and pH is negligible and non-stereoselective.

Adrenergic beta-Antagonists↗

Retention of small charged impurities during ultrafiltration.

Ultrafiltration is used to remove small impurities from a variety of processing streams. However, the clearance of small charged impurities may be inadequate due to electrostatic exclusion by the charged ultrafiltration membranes, an effect that has been largely unappreciated. Ultrafiltration experiments were performed to evaluate the transmission of several model impurities with different electrical charge through ultrafiltration membranes having different surface charge characteristics. Highly charged impurities are strongly rejected by charged cellulose and polyethersulfone membranes even though these solutes are much smaller than the membrane pore size. These effects could be eliminated by using high ionic strength solutions to shield the electrostatic interactions. The sieving data are in good agreement with model calculations based on the partitioning of charged spheres into charged cylindrical pores. Guidelines are developed for estimating conditions needed to obtain effective removal of small charged impurities through charged ultrafiltration membranes.

Buffers↗

Applications of pulsed ultrafiltration-mass spectrometry.

Pulsed ultrafiltration-mass spectrometry (PUF-MS) is a method with a variety of uses for the discovery and development of biologically active small molecules, including the screening of combinatorial libraries and natural product extracts for biologically active compounds, investigation of thermodynamic and kinetic ligand-receptor binding parameters, high-throughput metabolic screening, and the screening of combinatorial libraries and botanical extracts for electrophilic metabolites. Solution-phase ligand-screening assays that use pulsed ultrafiltration-mass spectrometry are useful for "reverse pharmacology" studies in which a macromolecular receptor of interest has been isolated, but ligands for the receptor are needed. Protein-binding studies that involve pulsed ultrafiltration can be used to rapidly determine classical binding parameters for interactions between a macromolecular receptor and a compound of interest. Metabolic screening assays can identify substrates for cytochromes p450, and should be capable of characterizing phase I metabolites with a throughput of at least 60 compounds/hr. Pulsed ultrafiltration can also be used in conjunction with LC-MS-MS to screen mixtures for compounds that might be activated metabolically to electrophilic quinoid and epoxide metabolites by cytochrome p450; that screening can provide early warning of compounds likely to be toxic when administered in large doses. The combination of pulsed-ultrafiltration extraction and mass spectrometric detection provides the sensitivity and selectivity necessary to characterize compounds present at low concentrations in complex chemical mixtures, and is applicable to the analysis of biologically active compounds from combinatorial libraries and botanical extracts.

Animals↗

An ultrafiltration method for the removal of interfering agents and its application to the determination of free ammonia in solutions of oxystarch by the Berthelot reaction method.

Oxystarch was chosen as a model compound for studying biological ammonia-sequestering systems. Ammonia was determined by use of an ion-selective electrode, by L-glutamate dehydrogenase (L-GDH), and by two different Berthelot procedures, in the presence and absence of oxystarch. In the presence of total oxystarch the Berthelot method, particularly when low concentration reagents were used, detected significantly less (P < 0.10) free ammonia than either L-GDH or ion-selective electrode methods. A 0.5-kDa molecular weight cutoff sample ultrafiltration step was added prior to analysis by L-GDH and Berthelot procedures. To facilitate complete removal of oxystarch by the ultrafiltration step, oxystarch was dialyzed before use, yielding a high-molecular-weight fraction (> 1 kDa). Removal of high-molecular-weight oxystarch species and bound ammonia by ultrafiltration of samples prior to assay completely negated discrepancies between ammonia levels measured by L-GDH and both Berthelot methods. The correlation of the levels of measured ammonia, as determined by L-GDH and Berthelot methods, in mixtures with high-molecular-weight oxystarch was significantly improved by the addition of the sample ultrafiltration step. Improved correlation of results from such fundamentally different methods demonstrates the removal of interfering agents as well as the nonperturbatory nature of the improved procedure. The addition of such an ultrafiltration step may be applied to the determination of ammonia by the otherwise interference-prone Berthelot assay in mixtures with any interfering macromolecules, without the inconvenience or potential variabilities associated with distillation or diffusion procedures.

Ammonia↗

A high-performance liquid chromatographic assay for CI-973, a new anticancer platinum diamine complex, in human plasma and urine ultrafiltrates.

CI-973 is a new platinum compound with antitumor properties that is currently undergoing phase II clinical trials. A high-performance liquid chromatographic (HPLC) assay was developed and validated for ultrafiltrates of human plasma and urine to support phase I clinical trials. Plasma ultrafiltrate (0.5 ml) was extracted using C18 solid-phase cartridges. Urine was diluted 10-fold and extracted first with SAX solid-phase cartridges and then with C18 cartridges. For both matrices, the eluate from the C18 cartridges was injected directly. A Whatman PAC 10 column (4.6 x 250 mm, 10-microns particle size) and ultraviolet detection at 205 nm were used for both analyses. The mobile-phase buffer was 0.05 M sodium perchlorate (pH 2.3). The mobile-phase acetonitrile:buffer ratio, column temperature, and flow rate were 89:11 (v/v), 40 degrees C, and 2.0 ml/min, respectively, for the plasma ultrafiltrate assay and 85:15 (v/v), 50 degrees C, and 1.0 ml/min, respectively, for the urine ultrafiltrate assay. Standard curves were linear from 0.25 to 500 micrograms/ml and from 1.0 to 250 micrograms/ml for the plasma and urine assays, respectively. The accuracy of the assay lay within 4.5% of the nominal values, and the precision was 6.2%; the recovery of CI-973 varied from 79.2% to 105%. CI-973 remains stable in plasma for at least 6 h, at room temperature, in ultrafiltrates of both matrices for at least 15 days at -72 degrees C, and in water for at least 6 months at -72 degrees C.

Antineoplastic Agents↗

Determination of the major zinc fractions in human serum by ultrafiltration.

In this paper we report a method for measuring ultrafiltrable zinc in human serum by electrothermal atomic absorption spectrophotometry. We show also that ultrafiltration permits to determine alpha-2-macroglobulin bound zinc and loosely bound zinc if a strong zinc ligand (EDTA) is added to serum before ultrafiltration. This last fraction, after deduction of ultrafiltrable zinc, represents roughly all albumin bound zinc. In 20 controls we found that ultrafiltrable zinc amounted 0.311 mumol/L (S.D. = 0.117 mumol/L), alpha-2 macroglobulin bound zinc 3.08 mumol/L (S.D. = 0.221 mumol/L), and albumin bound zinc 12.11 mumol/L (S.D. = 1.95 mumol/L). Our method needs only a small volume of serum, it is simple and rapid but also very accurate and reliable. The loosely bound fraction is very dynamic and, representing the physiologically active part of serum zinc, it could be a good marker of zinc deficiency.

Biomarkers↗

Profiling of uremic ultrafiltrate using high resolution gas chromatography-mass spectrometry - identification of 6 polyphenols.

An analytical method for separation and identification of compounds in uremic ultrafiltrate has been developed using a glass capillary column gas chromatography-mass spectrometry-computer system. The ultrafiltrate samples of blood were obtained during hemodialysis treatment of chronic uremic patients and non-uremic patients using the extracorporeal ultrafiltration method. Sample preparation consisted of acidification, extraction, evaporation, and trimethylsilylation. Many compounds were newly identified in the uremic ultrafiltrate by electron impact ionization, chemical ionization, and high resolution mass spectrometry. Six toxic polyphenols, namely, catechol, resorcinol, hydroquinone, 2-methoxyresorcinol, 3-methoxycatechol, and methoxyhydroquinone were first detected in the uremic ultrafiltrate.

Acute Kidney Injury↗

A simplified ultrafiltration method for determination of serum free cortisol.

We describe the suitability of the Amicon MPS-1 centrifugal ultrafiltration device and the YMB membrane for measuring free cortisol in serum. The method combines two independent assays: total cortisol and the ultrafiltrate fraction of added [3H]cortisol. The unbound fraction is determined in 0.25-0.30 ml of ultrafiltrate collected from 0.6 to 1 ml of serum that has been equilibrated with [3H]cortisol at 37 degrees C for 20 min. The assay is rapid (less than 1 h), practical ( no more than 0.6 ml of serum is necessary) and repeatable (CV: 3.8% within-assay and 12.2% in different assays). Error introduced in free cortisol measurement due to dilution effects in dialysis is systematically defined, and the effect of tracer purity on the ultrafiltration method is examined. Dialyzed sera from normal men and women, from patients with Cushing's disease and adrenal insufficiency, and from pregnant women gave ultrafiltration results that accurately duplicated those obtained by previous dialysis.

Adsorption↗

Free thyroxine measured in undiluted serum by dialysis and ultrafiltration: effects of non-thyroidal illness, and an acute load of salicylate or heparin.

In vitro dilution of serum during processing of a free T4 assay explains to some extent the divergent results obtained in non-thyroidal illness. If serum from such patients contains low affinity T4 protein binding inhibitors, as has been suggested, in vitro dilution will result in spuriously reduced serum free T4 measurements. If these inhibitors cross the dialysis membrane in an equilibrium dialysis assay, their inhibitory effect will be weakened, and in vitro free T4 levels will decrease, even in undiluted serum. In contrast, ultrafiltration methods on undiluted serum seem accurate. We have compared a new, commercially available dialysis technique with an in-house ultrafiltration method for free T4 measurements in undiluted serum. Control subjects (n = 41) had 14% higher free T4 (P < 0.02) by ultrafiltration. Non-thyroidally ill patients not receiving glucocorticoids or dopamine (n = 54) had unaltered free T4 levels, 28.4 +/- 10.3 pmol/l (dialysis) and 31.0 +/- 10.3 pmol/l (ultrafiltration). Dopamine infusion in somatic ill patients (n = 11) resulted in reduced free T4 in both assays but only significantly for dialysis, and subjects with familial dysalbuminemic hyperthyroxinemia (n = 8) had unaltered free T4 levels in both assays. Salicylate (1.5 g) given orally 09:00 h. (n = 5) resulted within 30 min, in increased (P < 0.01) free T4 as measured by both techniques, although more pronounced and sustained as measured by ultrafiltration. Serum TSH decreased concomitantly (P < 0.01). These findings were confirmed when salicylate was administered at 13:00 h. (n = 8). The dialysis procedure resulted in a decrease in serum salicylate of 14% (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Ultrafiltration and subsequent high performance liquid chromatography for in vivo determinations of the protein binding of etoposide.

Etoposide is extensively (approximately 94%) bound to plasma proteins and the free non-protein-bound levels have been shown to correlate more closely to toxicity than total drug concentrations. A rapid and easily performed method, compared to the time consuming equilibrium dialysis, to obtain the free fraction is needed. The aim of this study was to evaluate ultrafiltration and subsequent high performance liquid chromatography (HPLC) for the determination of protein binding of etoposide. Spiked plasma from healthy, drug-free volunteers was used to compare ultrafiltration, using Amicon Centrifree filters, with equilibrium dialysis at 37 degrees C. The variability (CV) of the ultrafiltration method was 6.1 and 13.5% (n = 6) at 37 degrees C and room temperature (RT), respectively. The relative size of the free fraction obtained by ultrafiltration at 37 degrees C and RT was 1.22 (P = 0.0005) and 0.37 (P = 0.0001), respectively, compared with equilibrium dialysis at 37 degrees C. The chromatographic separation of metabolites from the mother compound when free etoposide is analyzed is crucial. It is shown that a hydroxy-acid metabolite of etoposide is quite dominant in a protein-free plasma fraction. The free concentrations were determined throughout a dose interval of 24 h in a patient receiving etoposide 100 mg/m2 daily. Ultrafiltration and subsequent HPLC is considered convenient and suitable for in vivo pharmacokinetic investigations.

Antineoplastic Agents↗

Determination of the anticancer drug, 15-deoxyspergualin, in plasma ultrafiltrate by liquid chromatography and precolumn derivatization with naphthalene-2,3-dicarboxaldehyde/cyanide.

An alternative analytical method for the determination of 15-deoxyspergualin in plasma is described. The drug was initially separated from the plasma matrix by ultrafiltration and a precolumn derivatization step was performed with naphthalene-2,3-dicarboxaldehyde in the presence of sodium cyanide to yield the fluorescent N-substituted 1-cyanobenz[f]isoindole (CBI) derivative. The CBI derivative was separated and quantitated by reversed-phase chromatography using an ODS Hypersil column and mobile phase of KH2PO4 (0.1 M)-H3PO4-acetonitrile (48:0.8:52, v/v/v) containing dodecyl sodium sulphate (18 mM). The excitation and emission wavelengths for the fluorescence detector were 420 and 490 nm, respectively. The peak height was linearly related to drug concentration over the range from 5 ng ml-1 (10 nM) to 10 micrograms ml-1 (20 microM) in phosphate buffer (0.1 M, pH 7.0), spiked plasma ultrafiltrate and ultrafiltrate obtained from spiked plasma. Measurements could be made with a relative standard deviation of 4.5% or less in phosphate buffer (0.1 M, pH 7.0), 6.1% or less in spiked plasma ultrafiltrate and 12% or less in ultrafiltrate obtained from spiked plasma.

Antibiotics, Antineoplastic↗

Determination of endogenous ions in intercellular fluid using capillary ultrafiltration and microdialysis probes.

Capillary ultrafiltration probes are novel sampling tools for continuously monitoring small molecules in the extracellular fluid of awake animals. Capillary ultrafiltration uses a vacuum applied to hydrophilic membrane fibres and extracts intercellular fluid and quantitatively recover many small hydrophilic molecules. The effects of continuously removing a small amount of fluid from the interstitial space are not known. The concentration of sodium, potassium, calcium and inorganic phosphorus were determined in the collected ultrafiltrates from subcutaneous tissue. These values were compared to literature values and to concentrations determined for the same animals using microdialysis. The concentrations of sodium, potassium, calcium and inorganic phosphorous were found to be 140 +/- 4, 3.7 +/- 0.1, 1.1 +/- 0.1 and 1.7 +/- 0.1 mM, respectively, in the subcutaneous ultrafiltrates obtained from rats. These corresponded very well with literature values and microdialysates, obtained, using pure water as the perfusate, in subcutaneous tissue. The concentration of sodium and potassium were determined to be 142 +/- 2 mM and 3.6 +/- 0.2 mM, respectively, for the dialysates. Hyperinsulinemic-induced decrease in intercellular potassium levels under a euglycemic clamp were monitored using capillary ultrafiltration probes in rats to further validate this technique for monitoring small molecule dynamics in the intercellular space. The intercellular level of potassium in rats decreased from 3.6 +/- 0.5 to 2.6 +/- 0.3 mM after an acute dose of pork insulin.

Animals↗

Investigation of high-throughput ultrafiltration for the determination of an unbound compound in human plasma using liquid chromatography and tandem mass spectrometry with electrospray ionization.

A high-throughput ultrafiltration method with a direct injection assay has been developed to determine unbound concentrations of a high-protein binding compound, an alpha(v)beta(3) bone integrin antagonist (I), in human plasma for a clinical pharmacokinetic study. The 96-well MultiScreen filter plate with Ultracel-PPB membrane was evaluated for the separation of unbound from protein-bound compound I by ultrafiltration. The sample preparation was automated using a Packard MultiPROBE II EX liquid handling system to transfer the plasma samples to the 96-well PPB plate for centrifugation and to prepare ultrafiltrate samples for analysis. Using on-line extraction with a column-switching setup for sample clean-up and separation, the ultrafiltrate samples were directly injected onto a reversed-phase HPLC system and analyzed using a mass spectrometer interfaced with an electrospray ionization (ESI) source in the positive ionization mode (LC/ESI-MS/MS). The performance of the ultrafiltration using Ultracel-PPB 96-well plate for unbound I analysis was evaluated and optimized with respect to sample volume, centrifugation temperature, speed and time, and the relationship of the well positions of the PPB plate versus filtrate volumes and concentrations. The assay intraday accuracy and precision were between 93.9 and 104.8 and <7.3% (CV), respectively. The linear range of the calibration curve for the assay was 0.1-500 ng/mL on a Finnigan TSQ Quantum LC/ESI-MS/MS system. Evaluation and validation of the unbound plasma assay demonstrated it to be rapid, sensitive and reproducible.

Centrifugation↗

Protein binding of aspirin and salicylate measured by in vivo ultrafiltration.

OBJECTIVE: Methods for measuring protein binding of drugs generally require direct measurement of the concentration of unbound drug and thus may require a highly sensitive assay. In vivo ultrafiltration has been used to determine protein binding of endogenous substances. We have examined its value for measuring protein binding of drugs because it requires measurement of only the concentration of total drug, not unbound drug, in plasma. METHODS: The protein binding of aspirin and its metabolite salicylate was measured in 29 healthy subjects 20 minutes after a single oral dose of 600 mg soluble aspirin, by the new method, in vivo ultrafiltration, as well as by a standard method, in vitro ultracentrifugation. RESULTS: The data for salicylate were examined systematically to determine the optimal method of determining estimates of protein binding by in vivo ultrafiltration. Estimates of protein binding of salicylate were 81.7% +/- 10.1% (mean +/- SD) by the in vivo method and 81.6% +/- 11.3% by in vitro ultracentrifugation. Bland-Altman analysis of agreement showed that within-individual differences in percentage of protein binding determined by the two methods did not differ significantly from zero (mean difference, 0.07%; 95% confidence interval, -2.33 to +2.46). There was a highly significant correlation between estimates of protein binding by the two methods (r = 0.82; p = 0.001). Protein binding of aspirin was estimated of protein binding by the two methods (r = 0.82; p = 0.001). Protein binding of aspirin was estimated at 58.3% +/- 9.6% by in vivo ultrafiltration and could not be estimated by in vitro ultracentrifugation because the concentration of unbound aspirin in plasma was below the limit of detection for the assay. CONCLUSION: In vivo ultrafiltration can be used to measure protein binding of drugs and has potential advantages over conventional methods. A sensitive assay may not be required because the unbound drug need not be measured, measurement in vivo may maintain more physiologic conditions, and it may be useful in measuring protein binding of drugs that are degraded rapidly in vitro.

Adult↗