Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “FILTRATION”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 415 records · Page 23Linked to original sources
PubMed1999

Probability of filtration surgery in patients with open-angle glaucoma.

OBJECTIVE: To investigate the probability of undergoing filtration surgery in either 1 or both eyes in patients in whom open-angle glaucoma was newly diagnosed. METHODS AND DESIGN: A retrospective community-based study of 295 residents of Olmsted County, Minnesota, in whom open-angle glaucoma was newly diagnosed between January 1, 1965, and December 31, 1980, was performed. Kaplan-Meier methods were used to estimate the cumulative probability of undergoing filtration surgery during a 20-year period. RESULTS: At 20 years of follow-up, the Kaplan-Meier cumulative probability of undergoing filtration surgery in at least 1 eye was estimated to be 23% (95% confidence interval, 16%-30%), and in both eyes the estimate was 12% (95% confidence interval, 6%-17%). Patients with optic nerve damage at the time of diagnosis were more likely to undergo surgery than patients with elevated intraocular pressure but no damage (1 eye, 39% vs 15%; both eyes, 27% vs 5%). CONCLUSION: This retrospective study of a white population newly diagnosed as having and treated for open-angle glaucoma indicates that while most patients did not undergo filtration surgery in the course of glaucoma therapy, at least one third of those with glaucomatous damage at the time of diagnosis underwent filtration surgery.

Aged↗
PubMed2003

Scale-down of continuous filtration for rapid bioprocess design: Recovery and dewatering of protein precipitate suspensions.

The early specification of bioprocesses often has to be achieved with small (tens of millilitres) quantities of process material. If extensive process discovery is to be avoided at pilot or industrial scale, it is necessary that scale-down methods be created that not only examine the conditions of process stages but also allows production of realistic output streams (i.e., streams truly representative of the large scale). These output streams can then be used in the development of subsequent purification operations. The traditional approach to predicting filtration operations is via a bench-scale pressure filter using constant pressure tests to examine the effect of pressure on the filtrate flux rate and filter cake dewatering. Interpretation of the results into cake resistance at unit applied pressure (alpha) and compressibility (n) is used to predict the pressure profile required to maintain the filtrate flux rate at a constant predetermined value. This article reports on the operation of a continuous mode laboratory filter in such a way as to prepare filter cakes and filtrate similar to what may be achieved at the industrial scale. Analysis of the filtration rate profile indicated the filter cake to have changing properties (compressibility) with time. Using the insight gained from the new scale-down methodology gave predictions of the flux profile in a pilot-scale candle filter superior to those obtained from the traditional batch filter used for laboratory development.

Computer Simulation↗
PubMed1985

Subfractionation and characterization of native and incubation enlarged human plasma high density lipoprotein particles by high performance gel filtration.

Incubation of human plasma in vitro at 37 C results in an increase of the mean particle size of the high density lipoproteins (HDL) accompanied by an almost complete disappearance of the original particles present prior to incubation. A rapid high performance gel filtration technique has been developed in order to study the chemical composition of subfractions of native and incubation enlarged HDL particles as a function of particle size. Subfractionation of HDL isolated by preparative ultracentrifugation from 3 normal human plasmas incubated in vitro at 0 and 37 C for 24 hr have been performed using a 150 cm long TSK-G 3000 SW column. The separation time was less than 65 min. The curves obtained at high performance gel filtration of HDL, by monitoring the effluents from the column at 280 nm, agreed well both in positions of peak maxima and relative peak intensities with the particle distribution patterns observed at polyacrylamide gradient gel electrophoresis of the corresponding HDL preparations run in parallel. The different HDL particle subfractions of the effluents from the gel filtration column have been characterized by quantification of free and esterified cholesterol, total phospholipids and apolipoprotein A-I and A-II. The incubation enlarged HDL particles, subfractionated by the high performance gel filtration technique, were found to have a composition which differed from that of native HDL particles of corresponding size. Incubation enlarged HDL had a generally higher and almost constant relative cholesteryl ester content over the whole particle range compared to native HDL in which a continuous increase in relative cholesteryl ester content could be observed when going from large to small particles. The molar ratio of phospholipids to free cholesterol was higher in small native HDL particles than in the corresponding large ones. The relation between apolipoprotein A-I and A-II remained nearly constant between small and large HDL particles in each subfractionation experiment. The results demonstrate that the high performance gel filtration technique is a rapid and reproducible means for studying the composition of subfractions of HDL particle populations.

Antibodies↗
PubMed2005

Membrane fouling and dialysate flow pattern in an internal filtration-enhancing dialyzer.

For efficient removal of large molecular weight solutes by dialysis, several types of internal filtration-enhancing dialyzers (IFEDs) are commercially available. However, in a pressure-driven membrane separation process (i.e., filtration), membrane fouling caused by adhesion of plasma proteins is a severe problem. The objective of the present study is to investigate the effects of internal filtration on membrane fouling based on the membrane's pure-water permeability, diffusive permeability, and sieving coefficient. Hemodialysis experiments were performed with two different dialyzers, IFEDs and non-IFEDs. Local membrane fouling in each dialyzer was evaluated by measuring the pure-water permeability, the diffusive permeability, and the sieving coefficient of native membranes and membranes treated with bovine blood. The effects of packing ratio on dialysate flow pattern were also evaluated by measuring the time required for an ion tracer to reach electrodes placed in the dialyzers. In the IFED, membrane fouling caused by protein adhesion is increased because of enhanced internal filtration only at the early stage of dialysis, and this fouling tends to occur only near the dialysate outlet port. However, enhanced internal filtration has little effect on measured membrane transfer parameters.

Animals↗
PubMed1994

Improvements and short-term viability of mouse epididymal spermatozoa recovered through the Spermprep filtration method.

This study was designed to determine the effects of Sephadex filtration (SpermprepI method) on the separation of motile, morphologically normal, mouse epididymal spermatozoa and to study the viability of the recovered spermatozoa over a 3-h incubation period. Spermatozoa were harvested from the caudae epididymie (5 animals per run or replication; n=10) following bilateral testicular excision, after which they were incubated in 2-ml of Test-Yolk buffer (TYB) at 37 degrees C for 15-min. The specimens were then split into 2 1-ml aliquots, with Aliquot 1 as the control and Aliquot 2 as the filtered sample. The SpermprepI column was employed according to the manufacturer's specifications using TYB. During filtration (10-min), 2 different fractions were obtained: first 5-min (Sample 1) and second 5-min (Sample 2). The 2 fractions were evaluated and incubated at 37 degrees C and assessed for percentage of motility and grade of motility (0 to 4) every 30-min for 3-h. Filtration resulted in a significant improvement in the percentage and grade of motility (91.5% and 3.0 vs 76.5% and 2.5, respectively). The results indicate that filtration with the SpermprepI method improved the percentage and grade of motility (P<0.05) but not the percentage of normal morphology of the spermatozoa. In addition, the SpermprepI method enabled the recovery of 45% (8.3x10(6) spermatozoa recovered) of the total number of spermatozoa processed in the control aliquot (18.4x10(6) spermatozoa), which is consistent with previous observations. Most importantly, filtered spermatozoa incubated for 3-h showed a greater percentage and grade of motility than the control spermatozoa (63% and 1.66 vs 39% and 0.82, respectively. The SpermprepI filtration method selected a higher proportion of quality spermatozoa, which also displayed significant long-term motility (longevity) during in vitro incubation.

Journal Article↗
PubMed2005

Organization of the pronephric filtration apparatus in zebrafish requires Nephrin, Podocin and the FERM domain protein Mosaic eyes.

Podocytes are specialized cells of the kidney that form the blood filtration barrier in the kidney glomerulus. The barrier function of podocytes depends upon the development of specialized cell-cell adhesion complexes called slit-diaphragms that form between podocyte foot processes surrounding glomerular blood vessels. Failure of the slit-diaphragm to form results in leakage of high molecular weight proteins into the blood filtrate and urine, a condition called proteinuria. In this work, we test whether the zebrafish pronephros can be used as an assay system for the development of glomerular function with the goal of identifying novel components of the slit-diaphragm. We first characterized the function of the zebrafish homolog of Nephrin, the disease gene associated with the congenital nephritic syndrome of the Finnish type, and Podocin, the gene mutated in autosomal recessive steroid-resistant nephrotic syndrome. Zebrafish nephrin and podocin were specifically expressed in pronephric podocytes and required for the development of pronephric podocyte cell structure. Ultrastructurally, disruption of nephrin or podocin expression resulted in a loss of slit-diaphragms at 72 and 96 h post-fertilization and failure to form normal podocyte foot processes. We also find that expression of the band 4.1/FERM domain gene mosaic eyes in podocytes is required for proper formation of slit-diaphragm cell-cell junctions. A functional assay of glomerular filtration barrier revealed that absence of normal nephrin, podocin or mosaic eyes expression results in loss of glomerular filtration discrimination and aberrant passage of high molecular weight substances into the glomerular filtrate.

Amino Acid Sequence↗
PubMed1997

Late onset of sequential multifocal bleb leaks after glaucoma filtration surgery with 5-fluorouracil and mitomycin C.

PURPOSE: To describe the late onset of sequential multifocal bleb leaks as a postoperative complication after filtering surgery with antimetabolites. MATERIALS: Retrospectively, 385 consecutive eyes (304 patients) undergoing trabeculectomy with 5-flurouracil (5-FU) or mitomycin C (MMC) from 1989 to 1994 were reviewed. Eyes with filtration bleb leak occurring 6 months or more after trabeculectomy were analyzed, and clinical characteristics of the filtration bleb, response to treatment, and bleb histopathology from eyes undergoing bleb excision were analyzed. RESULTS: In seven (1.8%) of 385 consecutive eyes from 304 patients undergoing glaucoma filtration surgery with 5-FU or MMC, repetitive bleb leaks in different locations of the bleb were observed from 9 to 44 months (mean, 20.4 months) after the procedure. One hundred ninety-three eyes (50%) were treated with 5-FU and the remaining eyes, with MMC. All eyes had transparent, avascular, lobular, cystic blebs. Bleb leaks occurred in five eyes treated postoperatively with subconjunctival 5-FU and in two eyes in which MMC was used intraoperatively. Three eyes (all treated with 5-FU) required surgical excision, and four eyes healed with soft contact lens, cyanoacrylate glue, or intrableb injection of autologous blood. Histopathology of the bleb leak sites demonstrated focal epithelial thinning and interruption with subjacent hypocellularity and stromal collagen degeneration. CONCLUSION: Late sequential multifocal bleb leaks may occur after glaucoma filtration surgery with administration of antimetabolites (5-FU or MMC) and are associated with epithelial break-down, hypocellularity, and stromal collagen necrosis in the filtration bleb.

Aged↗
PubMed1996

Histopathologic and immunohistochemical analysis of the filtration bleb after unsuccessful glaucoma seton implantation.

PURPOSE: To analyze histopathologically and immunohistochemically the filtration bleb after unsuccessful glaucoma seton implantation. METHODS: A von Denffer implant and two Molteno implants that were nonfunctional at three months, at 11 months, and at five years after implantation, respectively, were compared by evaluating the adjacent bleb with light microscopy and a panel of 11 antibodies to epithelial, mesenchymal, and inflammatory cells. RESULTS: The wall of the filtration bleb three months after implantation consisted of loosely arranged collagenous connective tissue with relatively abundant fibroblasts labeled with monoclonal antibodies (MAbs) V9 and Vim 3B4 to vimentin. At 11 months and at five years, the collagenous layers had become increasingly thick and the fibroblasts scarce. All blebs showed metaplastic myofibroblasts surrounded by tenascin, as identified by MAb 1A4 to alpha-smooth muscle actin and TN2 to tenascin, respectively. One bleb was lined by a monolayer of cells that reacted with MAb CAM 5.2 and CY-90 to cytokeratin 8 and 18, respectively, and was apparently derived from the identically reacting proliferating corneal endothelial cells. Another bleb harbored many macrophages and foreign-body giant cells. Little evidence of chronic inflammation around the seton was detected. CONCLUSIONS: A glaucoma seton may induce several processes that potentially decrease filtration. In addition to formation of a collagenous cyst, presence of myofibroblasts suggests long-standing scar modulation that potentially leads to compaction of the filtration membrane. Furthermore, macrophages and corneal endothelial cells may invade the filtration bleb.

Adult↗
PubMed1994

The effect of long-term medical therapy on the outcome of filtration surgery.

Long-term medical therapy and laser trabeculoplasty have been implicated as risk factors in the outcome of glaucoma filtration surgery. A retrospective analysis was performed of patients undergoing primary trabeculectomy during three key periods: before use of beta-blockers and laser trabeculoplasty (1975 and 1976), after introduction of beta-blocker therapy but before use of the laser (1980), and after common use of beta-blockers and laser (1985 and 1986). A total of 150 patients were included in the analysis. Eyes treated with laser trabeculoplasty at some time before filtration surgery had a decreased probability of successful filtration surgery (intraocular pressure less than 21 mm Hg, 75% vs 91% at one year, P = .05; 50% vs 86% at five years, P = .01). It is unknown whether laser was detrimental to subsequent filtration surgery or merely acted as a selection factor in identifying patients who would have less successful surgical outcomes. Preoperative use of topical medication did not influence the outcome of surgery. Patients having bilateral filtration surgery had similar outcomes between fellow eyes despite the longer use of therapy in the second eye (mean of 1.8 years between operations). In the subgroup of eyes with low preoperative intraocular pressures (less than 21 mm Hg), surgery was successful in reducing pressure to a lower level in 70% of eyes at one year (preoperative pressure, 17.8 mm; one-year postoperative pressure, 13.3 mm, P = .007).

Adult↗
PubMed2001

Capillary filtration is reduced in lungs adapted to chronic heart failure: morphological and haemodynamic correlates.

OBJECTIVE: To determine pulmonary capillary filtration in experimental chronic heart failure and to investigate some morphological and haemodynamic mechanisms that could account for reduced filtration in lungs adapted to chronic heart failure. METHODS: We studied pulmonary capillary filtration, vascular resistances and morphology in lungs from guinea-pigs adapted to chronic heart failure. Heart failure was induced by banding of the ascending aorta (n=66) or sham control operation (n=78) in guinea-pigs which were studied at 150+/-8 days post-operation. RESULTS: Reduced cardiac output, increased systemic vascular resistance and LV end diastolic pressure and increased LV and RV weight:body weight ratio (all P<0.05) indicated chronic heart failure at 5 months following aortic banding in guinea-pigs. Lung weight was increased (61%, P<0.05) in heart failure compared with controls, but lung water content was reduced (5.5%, P<0.05), a reversal of the pattern seen acutely. Studies in isolated perfused lungs demonstrated a reduced capillary filtration coefficient (0. 018+/-0.003 vs. 0.003+/-0.002 ml min(-1)mmHg(-1)g(-1), P<0.001), increased arterial (61%) and venous resistance (50%) in heart failure lungs, P<0.05. Wall thickness:lumen ratio was increased in small (<250 microm) pulmonary arterioles (0.15+/-0.02 vs. 0.08+/-0. 01) and venules (0.06+/-0.005 vs. 0.04+/-0.002) in heart failure, P<0.01. Alveolar septal volume fractions (35.2+/-5.1 vs. 23.1+/-2.7) and septal:air-space volume ratios (60.5+/-13.6 vs. 31.9+/-5.3) were also increased in heart failure, P<0.05. CONCLUSIONS: Pulmonary adaptation to chronic heart failure is associated with vascular and alveolar remodelling that contributes to increased vascular resistance and reduced capillary filtration. These changes are likely to be important in mediating resistance to pulmonary oedema in chronic heart failure.

Adaptation, Physiological↗
PubMedDate not supplied

Role of Pseudomonas aeruginosa culture filtrates in the association, invasion, and cytotoxicity against cloned cells from murine corneal epithelium and KB cells.

PURPOSE: To clarify the effect of Pseudomonas aeruginosa culture filtrates on the association with, invasion into, and cytotoxicity against cloned cells from murine corneal epithelial cells and KB cells. METHODS: Simian virus 40-transformed murine corneal epithelial (MCE) cells were established. Murine corneal epithelial cells and KB cells were infected with a protease-positive strain, IID1117 (Pa IID1117), and a protease-negative strain, IID1130 (Pa IID1130) of P. aeruginosa, and then tested for association and invasion of Pa IID1117. The cytotoxicity test was performed by incubating the cells with culture filtrate. RESULTS: Association of Pa IID1117 with KB cells pretreated with Pa IID1130 was significantly promoted. After pretreatment with culture filtrate, invasion was more effective into MCE cells than into KB cells. When infecting bacteria (Pa IID1117) were pretreated with protease inhibitor, invasion of the bacteria into MCE cells and KB cells clearly decreased. The cellular damage induced by the culture filtrate of Pa IID1130 was greater than the damage by that of Pa IID1117. CONCLUSION: These results suggest that association of P. aeruginosa with MCE cells and KB cells was influenced by the culture filtrates other than proteases, and that invasion of P. aeruginosa into MCE cells and KB cells was promoted by protease.

Animals↗
PubMed1980

Determination of mulecular-weight distribution of aorta glycosaminoglycans by automated gel filtration.

This report describes an automated gel filtration procedure for estimation of molecular weights of glycosaminoglycans using Technicon sugar analyzer. A Sepharose CL-6B column was used for filtration. The method has been applied to determining the distribution of molecular weights of glycosaminoglycans from human aorta. The glycosaminoglycans were fractionated on a Dowex 1 (Cl-) column prior to gel filtration. On gel filtration, hyaluronic acid resolved into two components with estimated molecular weights of 38,000 and 8000. A molecular weight of 22,000 was estimated for heparan sulfate. Chondroitin 6-sulfate and dermatan sulfate eluted from the column in one peak, suggesting these two glycosaminoglycans have a similar molecular weight, 18,000. The procedure was found suitable for studying the gel filtration behavior of proteoglycans from bovine aorta and lung.

Animals↗
PubMed2003

Sephadex and sephadex ion-exchange filtration improves the quality and freezability of low-grade buffalo semen ejaculates.

The effect of sephadex and sephadex ion-exchange filtration on the improvement in quality and freezability of low-grade buffalo semen ejaculates was assessed. Two types of filtration columns were used: one containing only sephadex G-10 (FS) and the other sephadex G-10 along with ion-exchangers (diethyl amino ethane-52 (DEAE-52) cellulose and carboxy methyl-52 (CM-52) cellulose; FS+IE). Unfiltered samples served as controls. Semen ejaculates extended in Tris-citric acid (1:4) (n=16; initial motility 40-50%) were filtered at the rate of 1.5 ml/min under negative pressure at room temperature (28-30 degrees C). The mean recovery rate (%) of motile spermatozoa in the FS (85.9+/-1.51) and FS+IE (77.10+/-2.28) filtrates did not differ significantly. Percentages of sperm motility, normal acrosomes, and intact plasma membranes were highest (P<0.05) in FS+IE, intermediate (P<0.05) in FS and lowest (P<0.05) in controls at the three stages of cryopreservation (postfiltration final dilution, after equilibration, and after freezing). Mean sperm abnormalities were lowest (P<0.05) in the filtrates of FS+IE, moderate (P<0.05) in FS and highest in controls at all stages of freezing. Compared to dilution and equilibration, freezing greatly reduced (P<0.05) the overall percent motility, normal acrosomes and intact plasma membranes. The spermatozoa eluted through FS+IE columns proved more resistant (P<0.05) in bearing dilution, equilibration, freezing and thawing stresses than the spermatozoa from FS and control samples. It is concluded that filtration systems containing an FS+IE column can effectively enhance the quality and freezability of extended, low quality buffalo semen.

Acrosome↗
PubMed1998

Effect of column filtration upon the quality parameters of fresh dog semen.

We filtered dog semen through various resin columns to obtain a quick, simple system for improving semen quality. Fresh ejaculates were filtered through columns with either glasswool or a chemically-inert polypropylene network disc. The columns were filled with Sephadex G-15 (nonionic resin), Sephadex A-50 (anionic-exchange resin), Sephadex C-50 (cationic-exchange resin) or a combination of Sephadex A-50 and C-50. Filtration through glasswool improved semen quality, with a significant (P < 0.001) increase in the percentage of viability and decrease in the percentage of altered acrosomes (P < 0.001) and total abnormalities (P < 0.001). Total motility was not modified, but curvilinear velocity or linearity of the movement were improved using the glasswool bed. The effect of the glasswool was so intense that it masked the effects of the filtration resins. Substitution of glasswool by polypropylene discs resulted in an unmasking of the effects of the resins, although the polypropylene exerted slight effects on semen. Elution of the spermatozoa through Sephadex G-15 or Sephadex C-50 resulted in a decrease of altered acrosomes. However, filtration through Sephadex A-50 increased viability and decreased the percentage of altered acrosomes and total abnormalities. Combined filtration through Sephadex A-50 and C-50 yielded the combined results observed with the resins individually. Ultrastructural imaging of the interaction between spermatozoa and the beds and resins showed that the cells were loosely deposited upon the glasswool fibers and the Sephadex G-15 particles, whereas close interaction was observed between spermatozoa and Sephadex A-50 and C-50 particles. The whole of the sperm cell bound to C-50 particles, whereas spermatozoa were specifically bonded to A-50 particles in the apical region of the head and in segments of the tail, which were periodically distributed. The data suggest that filtration through glasswool or an anionic resin-exchange can significantly improve dog semen quality.

Acrosome↗
PubMed1993

Effects of intraoperative 5-fluorouracil or mitomycin C on glaucoma filtration surgery in the rabbit.

PURPOSE: To establish the effects of single intraoperative exposures to either 5-fluorouracil (5-FU) or two different concentrations of mitomycin C (MMC) on filtration surgery in the rabbit. METHODS: A prospective, randomized, masked-observer, placebo-controlled study was performed to compare the effects on filtration surgery in rabbits of a single 5-minute intraoperative exposure to 5-FU (50 mg/ml), MMC (0.2 mg/ml), or MMC (0.4 mg/ml) with control eyes treated with distilled water. RESULTS: Intraocular pressures (IOPs) remained lower for longer and bleb survival was increased (P < 0.05) with all three treatments compared with control eyes. The effect of 5-FU was more transient than MMC. Bleb survival was prolonged in the following order: MMC 0.4 > MMC 0.2 > 5-FU 50 mg/ml. At 30 days, blebs were present in 100% of eyes treated with MMC 0.4 mg/ml, 60% of eyes treated with MMC 0.2 mg/ml, and 0% of eyes treated with 5-FU 50 mg/ml or distilled water. The blebs in the eyes treated with MMC were thinner, and significant complications (endophthalmitis, transient corneal opacification and neovascularization, and a presumed bleb leak) only occurred in the eyes treated with MMC 0.4 mg/ml. CONCLUSIONS: The authors' results suggest that 5-FU and MMC at these concentrations, delivered as a single intraoperative dose, prolong the survival of filtration surgery for different lengths of time in the rabbit, which is a model that normally exhibits aggressive healing and rapid failure of filtration surgery. The implications of these potentially titratable effects for filtration surgery in different categories of patients are discussed.

Animals↗
PubMed1998

Radio-iodination of neurotrophins and their delivery in vivo: advantages of membrane filtration and the use of disposable syringes.

This paper reports two simple improvements for the radio-iodination of neurotrophins and their delivery in vivo. (1) Neurotrophins can be effectively separated from free iodide by using membrane filtration devices. Seven methods for the separation of free iodide are compared, including dialysis, gel filtration, and membrane filtration. Membrane filtration of the iodinated protein has several important advantages over dialysis or gel filtration. These include the precise control over the final concentration; excellent recovery of the neurotrophin; easy and inexpensive procedure; performance of the entire procedure in a fume hood; and reduced volume of radioactive waste. (2) Disposable, inexpensive syringes are suitable for the delivery of small volumes of radio-iodinated or non-radioactive neurotrophins. Plastic disposable insulin syringes are compared with Hamilton syringes. The ejection volume of the disposable syringes is surprisingly reliable in the dose range 2-15 microl. Their in vivo performance was tested by injections in the eyes of chick embryos in ovo. The amounts remaining in the eye varied significantly less with the disposable syringes. Leakage into the surrounding eye-muscles after intraocular injection was significantly more frequent with Hamilton syringes than with the disposable syringes. Thus, disposable syringes can be a reliable and cost-effective alternative for drug delivery of 2-15 microl volumes.

Animals↗
PubMed2002

Single-step extraction of fluconazole from plasma by ultra-filtration for the measurement of its free concentration by high performance liquid chromatography.

High performance liquid chromatography (HPLC) is the reference method for measuring concentrations of antimicrobials in blood. This technique requires careful sample preparation. Protocols using organic solvents and/or solid extraction phases are time consuming and entail several manipulations, which can lead to partial loss of the determined compound and increased analytical variability. Moreover, to obtain sufficient material for analysis, at least 1 ml of plasma is required. This constraint makes it difficult to determine drug levels when blood sample volumes are limited. However, drugs with low plasma-protein binding can be reliably extracted from plasma by ultra-filtration with a minimal loss due to the protein-bound fraction. This study validated a single-step ultra-filtration method for extracting fluconazole (FLC), a first-line antifungal agent with a weak plasma-protein binding, from plasma to determine its concentration by HPLC. Spiked FLC standards and unknowns were prepared in human and rat plasma. Samples (240 microl) were transferred into disposable microtube filtration units containing cellulose or polysulfone filters with a 5 kDa cut-off. After centrifugation for 60 min at 15000g, FLC concentrations were measured by direct injection of the filtrate into the HPLC. Using cellulose filters, low molecular weight proteins were eluted early in the chromatogram and well separated from FLC that eluted at 8.40 min as a sharp single peak. In contrast, with polysulfone filters several additional peaks interfering with the FLC peak were observed. Moreover, the FLC recovery using cellulose filters compared to polysulfone filters was higher and had a better reproducibility. Cellulose filters were therefore used for the subsequent validation procedure. The quantification limit was 0.195 mgl(-1). Standard curves with a quadratic regression coefficient > or = 0.9999 were obtained in the concentration range of 0.195-100 mgl(-1). The inter and intra-run accuracies and precisions over the clinically relevant concentration range, 1.875-60 mgl(-1), fell well within the +/-15% variation recommended by the current guidelines for the validation of analytical methods. Furthermore, no analytical interference was observed with commonly used antibiotics, antifungals, antivirals and immunosuppressive agents. Ultra-filtration of plasma with cellulose filters permits the extraction of FLC from small volumes (240 microl). The determination of FLC concentrations by HPLC after this single-step procedure is selective, precise and accurate.

Animals↗
PubMed1998

IgE-sensitization to cellular and culture filtrates of fungal extracts in patients with atopic dermatitis.

BACKGROUND: Patients with atopic dermatitis may experience exacerbations of eczema triggered by various inflammatory stimuli. One mechanism may be IgE-mediated reactions to dermatophytes since these patients are more likely to acquire skin infections with dermatophytes and may become sensitized. OBJECTIVE: This study investigates IgE-sensitization to fungi in patients with atopic dermatitis and compares the biologic activity of culture filtrates and cellular fungal extracts. The following allergen extracts were provided as culture filtrates and cellular extracts: Candida albicans, Fusarium moniliforme, and Penicillium notatum. In addition, Pityrosporum ovale and Trichophyton rubrum cultures were included in the test panel. METHODS: Fifteen patients with clinical findings suggesting dermatophytosis and 11 controls were selected. Each subject was tested by leukocyte histamine release and skin prick test to each fungal extract. The extracts were separated and reduced by sodium dodecylsulfate polyacrylamide gel electrophoresis and analyzed by IgE-immunoblotting with sera from all study subjects. RESULTS: Fourteen patients (93%) reacted to one or several fungal extracts by releasing histamine when challenged in vitro. By immunoblotting experiments, patient sera showed binding to a wide range of components in all extracts. Patient sera recognized allergenic components shared by culture filtrates and cellular extracts but with higher frequent and greater intensity in culture filtrates. Although culture filtrates generated more frequent and potent IgE-reactions than the cellular extracts, the difference was not statistically significant. Biologic potency was similar when evaluated by skin prick tests and leukocyte histamine release. CONCLUSION: Patients with atopic dermatitis may develop specific IgE-antibodies to a number of fungi as demonstrated by IgE-immunoblotting. In selected patients, fungi may trigger an IgE-mediated reaction that may contribute to the exacerbation of eczema. Approximately, one-half of the patients, however, produced IgE-antibodies to fungal (glyco)proteins without a significant histamine release or skin test response possibly because of nonspecific interaction with carbohydrate moieties on IgE and poor biologic activity of IgE antibodies directed to cross-reactive carbohydrate determinants of fungal glycoproteins. This warrants caution when interpreting clinical relevance of serologic measurements of fungal IgE-antibodies.

Adolescent↗