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Carboplatin is a chemotherapy drug used in a variety of cancers with the primary toxicity being exposure-dependant myelosuppression. We present the development and validation of a simple, robust inductively coupled plasma mass spectrometry (ICP-MS) method to measure carboplatin in plasma ultrafiltrate. Plasma ultrafiltrates samples were prepared using Amicon Ultra 30,000 da cut-off filters and then diluted with ammonia EDTA before ICP-MS analysis. The assay was validated in the range 0.19–47.5 mg/L carboplatin in ultrafiltrate. The assay was linear (r 2 > 0.9999), accurate (<6% bias, 12% bias at LLOQ) and precise (intra- and inter-day precision of <3% coefficient of variation). No matrix effects were observed between plasma ultrafiltrate and aqueous platinum calibrators and recovery was complete. The assay was applied to 10 clinical samples from patients receiving carboplatin. Incurred sample reanalysis showed reproducible values over 3 analysis days (<6% CV). As plasma stability prior to ultrafiltration has been a major concern in previous clinical studies this was studied extensively at room temperature (22 °C) over 24 h. Carboplatin was found to be stable in both spiked plasma (n = 3) and real patient samples (n = 10) at room temperature for up to 8 h before ultrafiltration. This makes routine measurement of carboplatin concentrations in clinical settings feasible. [ABSTRACT FROM AUTHOR]

Vacuum evaporation is performed to concentrate milk constituents before their spray-drying. During this step, the mineral fraction and especially calcium phosphate precipitates. The aim of this study was to evaluate the mineral behavior with a special attention paid on calcium and inorganic phosphate during vacuum evaporation of milk ultrafiltrate (UF) and milk microfiltrate (MF)). Water evaporation was performed with a pilot-scale falling-film evaporator with a configuration close to those used in industry. Samples were analyzed for levels of water evaporation. Different approaches were used like i. analyses of mineral contents and their precipitation; ii. theoretical calculation of salt formed and ion distribution. During evaporation, formation of trouble with presence of particles was determined. In the same time, increases of all components present were determined with a precipitation of calcium phosphate. With UF, deposits of calcium phosphate in the evaporator were deduced whereas with MF, interaction of calcium phosphate with whey proteins limited the calcium phosphate precipitation. The mineral precipitates were always negatively charged. Moreover and due to evaporation, the increase in the ionic strength significantly decreased the negative charge present at the surface of calcium phosphate precipitates. [ABSTRACT FROM AUTHOR]

This study comprehensively investigated the influences of salinity, exposure concentration and time on the aggregate size, surface charge and dissolution of zinc oxide nanoparticles (ZnO–NPs; 20 nm) in seawater, and examined the interacting effect of salinity and waterborne exposure of ZnO–NPs on the marine diatom Thalassiosira pseudonana for 96 h. We found that aggregate sizes of ZnO–NPs significantly increased with increasing salinity, but generally decreased with increasing exposure concentration. Ion release decreased with increasing salinity, whereas the surface charge of the particles was not affected by salinity. The increased aggregate size and decreased ion release with increasing salinity, and consequently lower concentration of bioavailable zinc ions, resulted in decreased toxicity of ZnO–NPs at higher salinity in general in terms of growth inhibition (IC50) and chlorophyll fluorescence (EC50 – Ф Po and EC50 – Ф 2 ). However, IC50s and EC50s of ZnO–NPs were smaller than those of Zn 2+ (from ZnO–NPs ultrafiltrate and ZnCl 2 ), indicating that dissolved Zn 2+ can only partially explain the toxicity of ZnO–NPs. SEM images showed that ZnO–NPs attached on the diatom frustule surface, suggesting that the interaction between the nanoparticles and the cell surface may acerbate the toxicity of ZnO–NPs. Our results linked the physicochemical characteristics of ZnO–NPs in seawater with their toxicities to the marine diatom and highlighted the importance of salinity as an influential environmental factor governing the aggregation, dissolution and the toxicity of ZnO–NPs. [ABSTRACT FROM AUTHOR]

Abstract: A method for the determination of lopinavir (LPV) concentrations in cerebral spinal fluid (CSF) and plasma ultrafiltrate (UF) was developed and validated to analyze clinical specimens from patients receiving antiretroviral treatment with lopinavir/ritonavir. The CSF (400μL sample volume) final calibration range for LPV was 0.313–25.0ng/mL. The final calibration range for UF (50μL sample volume) was 1.25–100ng/mL. The samples were prepared using liquid–liquid extraction, concentrated, and analyzed using a reversed phase isocratic separation. Detection was achieved in positive mixed reaction monitoring mode on a triple quadrupole mass spectrometer. Isolation of LPV through chromatographic separation and proper selection of calibration matrix were important factors in achieving accurate results. Plasma UF was found to be an equivalent calibration matrix to CSF whereas plasma matrix produced a positive bias in samples with unknown concentrations. Artificial CSF media prepared chemically were biased and less superior than UF. Sources of plasma for the UF did not affect accuracy. Several CSF sources were tested for specificity of the method and LPV concentrations were accurately produced with atmospheric pressure chemical ionization source producing more accurate results than the electrospray source. The method successfully measured LPV concentrations in CSF that were previously undetectable by HPLC as well as UF from protein binding studies. [Copyright &y& Elsevier]

Introduction: Sepsis is a major cause of morbidity and mortality in critically ill patients. Sepsis is associated with cell necrosis and apoptosis. Circulating plasma levels of DNA have been found in conditions associated with cell death, including sepsis, pregnancy, stroke, myocardial infarction and trauma. Plasma DNA can also derive from bacteria. We have recently implemented a method to detect bacterial DNA and, in the present study, we validated this technique comparing it to standard blood culture in terms of diagnostic efficacy. Methods: We examined a cohort of 9 critically ill patients with a diagnosis of severe sepsis and acute renal failure requiring continuous renal replacement therapy (CRRT). We analyzed bacterial DNA in blood, hemofilters, and ultrafiltrate (UF) by polymerase chain reaction amplification of 16S rRNA gene sequence analysis. Standard blood cultures were performed for all patients. Results: The blood cultures from 2 of the 9 (22%) patients were positive. However, bacterial DNA was identified in the blood of 6 patients (67%), including the 2 septic patients with positive blood cultures. In 9 (100%) patients bacterial DNA was found on the filter blood side, whereas in 7 (78%) subjects it was found in the dialysate compartment of the hemofilters. Bacterial DNA was never detected in the UF. Conclusions: Using the 16S rRNA gene, the detection of bacterial DNA in blood and adsorbed within the filter could be a useful screening tool in clinically septic, blood culture-negative patients undergoing CRRT. However, the identification of the etiologic agent is not feasible with this technique because specific primers for the defined bacteria must be used to further identify the suspected pathogenic organisms. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background. Patients on chronic hemodialysis are likely to develop secondary hyperoxalemia. It is, however, difficult to measure plasma oxalate levels. To measure plasma oxalate levels, rapid plasma separation, deproteinization, and acidification are essential in preventing the formation of oxalate and the deposition of calcium oxalate within the test tube. The present study was undertaken to examine whether the oxalate level in dialyzer ultrafiltrate is potentially useful for estimating plasma oxalate levels. Methods. In nine patients on chronic hemodialysis, the plasma, after deproteinization with a filter, and the ultrafiltrate from the dialyzer before hemodialysis were acidified to a pH level of less than 3, followed by the measurement of oxalate levels by ion chromatography. Also, oxalate levels were compared between acidified and nonacidified ultrafiltrates from the dialyzer. In the second part of the study, seven patients on chronic hemodialysis receiving erythropoietin therapy, in whom the ferritin level was more than 300 ng/ml and transferrin saturation was less than 25%, were intravenously administered ascorbic acid, 100 mg, three times a week, after each dialysis session to facilitate the utilization of stored iron. This treatment was continued until the serum ferritin level decreased to a level below 300 ng/ml (for 3 months, at a maximum). The oxalate level in the dialyzer ultrafiltrate after this treatment was compared with that before treatment. Results. The mean ± SE oxalate level in the dialyzer ultrafiltrate was 45 ± 6 µmol/l, essentially equal to the plasma oxalate level (46 ± 7 µmol/l). The plasma oxalate level had a significant positive correlation with the dialyzer ultrafiltrate oxalate level (plasma oxalate level = 0.99 × dialyzer ultrafiltrate oxalate level + 1.5; r = 0.95; P < 0.0001). The oxalate level in the acidified ultrafiltrate (45 ± µmol/l) did not differ significantly from that in the non-acidified ultrafiltrate (45 ± µmol/l). The mean ± SE duration of ascorbic acid administration was 64 ± 13 days. The hemoglobin level remained unchanged at 9.6 ± 0.4 g/dl, whereas the serum iron level increased significantly, from 34 ± 2 µg/dl to 43 ± 4 µg/dl (P < 0.05), and serum ferritin levels decreased significantly, from 645 ± 219 ng/ml to 231 ± 30 ng/ml after the treatment (P < 0.05). The oxalate level in the acidified ultrafiltrate showed no significant change after ascorbic acid administration (31 ± 8 µmol/l vs 47 ± 7 µmol/l). Conclusions. In patients on chronic hemodialysis, the oxalate level in acidified ultrafiltrate from the dialyzer was found to be useful for estimating the plasma level of nonprotein-bound oxalate. When administering ascorbic acid to hemodialysis patients, the plasma oxalate level can be monitored using this method. [ABSTRACT FROM AUTHOR]

Abstract: In metabolic acidosis, the concentrations of anions associated with intermediary metabolism are increased and can make a significant contribution to the observed acidosis. Here we describe a method for the rapid determination of the plasma ultrafiltrate profile of these anions using liquid chromatography coupled to electrospray ionisation mass spectrometry (LC/ESI-MS). The ultrafiltrate from patients with acidosis resulting from various causes were examined and the results compared to control values. Using the LC/ESI-MS method described, a unique plasma ultrafiltrate anion profile was obtained for each of the groups studied that provides rapid diagnosis of the type of underlying acidosis. [Copyright &y& Elsevier]

Abstract: The estimation of the uncertainty associated to the analytical methods is necessary in order to establish the comparability of results. Methods of Pt determination in biological fluids lack very often of information about uncertainty of results, with likely implications when results are used to interpret the mechanism of action of platinum compound or when they are considered to optimise the clinical therapies. An inductively coupled plasma-mass spectrometer (ICP-MS) method for the determination of Pt in biological fluids (plasma, ultrafiltrate and urine) of patients treated with antitumor agents has been developed and validated. The limits of quantification (LOQ) in the three matrices were 1.0, 0.1, and 2.0 μg/l, respectively. Intraday and interday precisions and accuracies were in good agreement with the FDA criteria for the validation of analytical methods. The validation study was implemented by assessing the uncertainty evaluation for Pt determination in the different matrices according to EURACHEM/CITAC Guide. [Copyright &y& Elsevier]

Abstract Background Apoptosis of polymorphonuclear leucocytes (PMNLs) is important for the resolution of inflammation. Recently, we demonstrated that glucose-modified proteins increase PMNL apoptosis. No protein factors in sera of uraemic patients attenuating PMNL apoptosis have been identified to date. Materials and methods We tested the influence of commercially available monoclonal immunoglobulin light chains (IgLCs) from multiple myeloma patients and polyclonal IgLCs isolated from haemodialysis patients, previously shown to modulate PMNL functions and to contribute to their prestimulation, on PMNL apoptosis. We detected morphological changes, DNA strand breaks and the loss of DNA content. Results All three apoptosis assays showed that κ and λ type IgLCs increase the percentage of viable PMNLs by inhibiting apoptosis in a concentration-dependent manner. The effect of IgLCs was abolished by specific antibodies. Addition of genistein abolished the reduction of PMNL apoptosis by IgLCs, suggesting that IgLCs exert their effect via tyrosine phosphorylation. Furthermore, we showed that the inhibition of caspase-3 activity is involved in the decrease of PMNL apoptosis. Conclusion In concentrations present in sera of uraemic patients IgLCs could interfere with the normal resolution of inflammation and thereby contribute to the chronic inflammatory state found in end-stage renal disease patients. [ABSTRACT FROM AUTHOR]

Effects of uremic ultrafiltrate on the regulation of the parathyroid cell cycle by calcitriol. Background. Calcitriol (CTR) is used in the treatment of hyperparathyroidism secondary to renal failure because it decreases parathyroid hormone (PTH) synthesis and parathyroid cell proliferation. Previous studies in tissues other than parathyroids have demonstrated that uremic factors affect the action of CTR on the target cells. We questioned whether the uremic milieu interferes with the inhibition of parathyroid cell proliferation by CTR. Methods. Studies were performed in vitro using freshly excised normal dog parathyroid tissue incubated for 24 hours with and without CTR and in the presence of either total uremic ultrafiltrate (UUF) from uremic patients or high-pressure liquid chromatography (HPLC)-derived fractions (hydrophilic compounds eluting early and hydrophobic compounds eluting late) of this UUF (F1 to F4). Parathyroid cell proliferation was assessed by flow cytometry. Results. The addition of CTR 10-8 and 10-7 mol/L to parathyroid tissue produced an inhibition of the proliferation that was prevented in the presence of UUF. In a medium containing CTR 10-8 mol/L, the addition of F1, F2 and F3, but not F4, prevented the CTR-induced inhibition of parathyroid cell proliferation. With CTR 10-7 mol/L, the inhibition of proliferation was observed even in the presence of F1, F2 and also F4, but was prevented by F3. Uric acid (7 mg/dL), indoxyl sulfate (5 mg/dL) and p-cresol (1.4 mg/dL), which coeluted with F1, F2 and F4, respectively, did not interfere with the inhibitory action of CTR 10-7 mol/L; however, the addition of phenol (0.14 mg/dL), which coeluted with F3, prevented the CTR-induced inhibition of parathyroid cell proliferation. Conclusions. The presence of uremic toxins prevents the inhibition of parathyroid cell proliferation induced by calcitriol. [ABSTRACT FROM AUTHOR]

Hemofiltration reduces the serum priming activity on neutrophil chemiluminescence in septic patients. Background. Priming of the polymorphonuclear neutrophil (PMN) response has been implicated in the activation of oxidative burst and tissue injury in patients with septic shock and acute renal failure (ARF). This study evaluated whether hemofiltration (HF) removes substances able to enhance the oxidative burst of PMNs. Methods. Chemiluminescence (CL) priming activity induced by sera and ultrafiltrates of seven patients with septic shock, multiorgan dysfunction syndrome, and ARF (ARF/HF group) and of 10 uremic stable patients (Control/HF group) was evaluated on normal human PMNs stimulated with bacterial formyl-methionyl-leucyl-phenylalanine (FMLP). Patients submitted to HF were studied by determining blood and ultrafiltrate interleukin-8 (IL-8), platelet-activating factor (PAF), and CL priming activity at the beginning (T0), and after four hours (T4) of treatment. Results. Preincubation of normal human PMNs with sera and ultrafiltrates from septic patients induced a potent priming of CL activity in subsequent FMLP stimulation. In the ARF/HF group, the prefilter blood concentrations of IL-8 and CL PMN-priming activity significantly decreased during the four hours of HF treatment, with a loss of IL-8 in the ultrafiltrate of 6930 (median, range 4292 to 9282) ng per four hours. PAF detected in the ultrafiltrate and associated with the membrane (7.3 ng, range 1.45 to 9.89) was minimal. In the ARF/HF group, a significantly positive correlation between CL PMN-priming activity and IL-8 concentrations was observed. The CL priming activity in blood and ultrafiltrates was reduced to 55 and 46% by preabsorption with monoclonal antibody (mAb) anti-IL-8. In contrast, the PAF receptor antagonist WEB 2170 did not affect CL priming activity. In the control/HF group, the CL PMN-priming activity was significantly lower than in the ARF/HF group and was... [ABSTRACT FROM AUTHOR]

It may be desirable to collect compounds directly from sites of interest if blood concentrations do not reflect tissue levels. Ultrafiltration and microdialysis probes may be used to do this, but the hollow fibers of these probes are quite fragile. For this reason, we developed a pull-through technique that allows their implantation into the ovine quadriceps muscle and femur.The sheep is placed under anesthesia in lateral recumbency. An incision is made midway between the patella and greater trochanter directly over the lateral femur. A hand drill is used to make a 4.5-mm hole into the medullary cavity through the lateral cortex of the distal femur. A second incision is then made over the greater trochanter. The drill bit is inserted into the trochanteric fossa and a hole is drilled distally through the medullary cavity of the femur to the level of the first hole. A looped 20-gauge wire is then inserted into the femur and removed through the distal hole. Suture is attached, and the wire is withdrawn, leaving the suture in place. The suture is tied to the ultrafiltration probe tubing, allowing the probe to be carefully drawn into position. For implantation into the muscle, a 10-gauge introducer is used. The introducer is placed through the quadriceps muscle and the probe is then threaded through it. This technique has been successfully performed on 18 sheep. All sheep tolerated the procedure well. Up to 2.0 mL/day of interstitial fluid was recovered from each site. The average lifetimes of the bone and muscle probes were 35 and 40 days, respectively. [ABSTRACT FROM AUTHOR]

Adsorption in hemodialysis. The use of sorbents in different blood purification techniques is reviewed. The sorbents used in these therapies are divided into two groups: ( 1 ) Adsorption occurs fundamentally because of the hydrophobic properties of the sorbents. In this group, the sorbents used in different dialysis techniques are charcoal and nonionic macroporous resins. ( 2 ) Adsorption occurs by chemical affinity, such as ion exchange resins and chemisorbents. Sorbents were initially used in hemoperfusion, which caused many adverse events; later, with the use of coated charcoal, these undesired effects decreased or disappeared, but the adsorptive properties, water control, and acid-base balance still created problems. For these reasons, the use of sorbents in the treatment of chronic renal failure was almost totally discontinued. Little by little, interest in these substances has reappeared, and at present, they have been used in combination with other blood purification techniques such as hemodialysis, hemofiltration, peritoneal dialysis, and finally, hemodiafiltration. Within the various hemodiafiltration techniques, paired filtration dialysis-charcoal is being used to regenerate the ultrafiltrate, which is used as the replacement fluid. Charcoal regenerates the ultrafiltrate and transforms it into a physiological solution with a normal electrolyte composition, calcium, bicarbonate, and glucose, having eliminated the majority of both middle and large molecule uremic toxins. If regeneration is done properly, this replacement fluid is bacteria and endotoxin free. Studies currently are underway on the adsorption of different inflammatory substances in the ultrafiltrate, which could lead to improvement in the biocompatibility of the system. [ABSTRACT FROM AUTHOR]