Ultrafiltrate Of Plasma Research Articles

Proteomic analysis of human plasma: failure of centrifugal ultrafiltration to remove albumin and other high molecular weight proteins.

Determination of specific low abundance proteins, usually by radiolabelled or enzyme-linked immunoassays in serum or plasma is widely used in diagnostic medicine. Substitution of these assays by a proteomic approach has been suggested, but this methodology has far from realised its potential as a diagnostic tool. The main protein fractions of plasma represent more than 80% of total protein, making the hundreds or even thousands of other proteins difficult to detect by two-dimensional electrophoresis (2-DE). Thus, loading sufficient sample to detect trace proteins invariably means excessive loading of albumin and other high abundance proteins. The aim of this study was to determine whether centrifugal ultrafiltration of whole plasma could be used to eliminate proteins exceeding a desired molecular weight cut-off. Cellulose filters with a 30 kDa molecular weight cut-off were used with whole plasma, and total protein was determined before and after ultrafiltration. Samples were processed by routine methods for 2-DE using 18 cm, pH 3-10 isoelectric focusing strips for the first dimension and 7-15% gradient gels for the second dimension followed by silver staining. Gel analysis of the retentate fraction (> 30 kDa) revealed a typical 2-DE plasma profile with most of the major landmark proteins in place and as expected, the gels lacked many of the smaller (< 30 kDa) proteins. Comparison with gels of the filtrate fraction (> 30 kDa) revealed very little difference. Not only were many of the higher molecular weight proteins still present, but some of the smaller < 30 kDa landmark proteins were absent. Overall, gels of both the retentate and filtrate fractions were less informative than gels of whole plasma.

Renal cortical vasoconstriction contributes to development of salt-sensitive hypertension after angiotensin II exposure.

Rats that are administered angiotensin II (AngII) for 2 wk develop persistent salt-sensitive hypertension, which can be prevented by the immunosuppressor mycophenolate mofetil (MMF) given during the AngII infusion. This study examined the contribution of glomerular hemodynamics (GFR dynamics) in the post-AngII hypertensive response to a high-salt diet (HSD) and the effect of MMF treatment. During AngII administration, rats developed severe hypertension (systolic BP [SBP], 185 +/- 3.9 mmHg), proteinuria, afferent and efferent vasoconstriction, and glomerular hypertension. Rats that received AngII+MMF showed similar responses to AngII; however, they developed lower proteinuria (P < 0.05). At 2 wk, AngII was withdrawn and SBP returned toward normal. Rats were then placed on an HSD (4% NaCl), resulting in a progressive increase in SBP (155 +/- 8.2 mmHg at week 1 and 163 +/- 4.5 mmHg at week 5). GFR dynamic alterations persisted after AngII was stopped, i.e., afferent and efferent vasoconstriction, decreased glomerular plasma flow and single-nephron GFR, and lower ultrafiltration coefficient. These changes correlated with the thickening of the afferent arteriole and with focal tubulointerstitial injury. In the AngII+MMF group, SBP remained unchanged throughout the HSD period (146 +/- 2.3 mmHg at week 1 and 148 +/- 4.4 mmHg at week 5) in association with less afferent arteriolar thickening and tubulointerstitial injury. Single-nephron GFR, glomerular plasma flow, efferent resistance, and ultrafiltration coefficient returned to normal with a significant reduction in afferent resistance. These results suggest a critical role of cortical vasoconstriction in salt-sensitive hypertension. The MMF-induced prevention of these changes suggests that immune mechanisms are involved in the vasoconstrictive response.

Determination of unbound concentration of the novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid in human plasma by ultrafiltration followed by high-performance liquid chromatography with fluorimetric detection

The novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a highly protein bound drug with narrow therapeutic window. We report a simple HPLC method with fluorimetric detection for the determination of free DMXAA concentration in human plasma. Sample preparation involves the ultrafiltration of plasma by a Centrisart device for 30 min at 2000 g and extraction with acetonitrile: methanol mixture. The method was validated with respect to recovery, selectivity, linearity, precision, and accuracy. Calibration curves for DMXAA were constructed at the concentration range of 0.5–40 μ M in blank plasma and phosphate buffer. The difference between the theoretical and calculated concentration and the relative standard deviation were less than 10% at all quality control (QC) concentrations. The HPLC method has been used for the analysis of preclinical studies.

Serous Body Cavity Fluid Examination

The serous body cavities are mesothelial-lined potential spaces that surround the lungs (pleura), heart (pericardium), and the abdomen and pelvis (peritoneum). Normally, movement of these organs is facilitated by a small amount of fluid, an ultrafiltrate of plasma. When production and resorption of this plasma ultrafiltrate are not properly balanced, fluid may accumulate within 1 or more serous cavities, resulting in a true fluid-filled cavity (effusion).1

THE EFFECT OF COLCHICINE ON PROXIMAL TUBULAR REABSORPTION

Aquaporins, expressed in the brush border membrane (BB) could play a pivotal role in glomerulo-tubular balance (GTB) by effecting adaptive changes of water permeability to the variations in the load of filtered solutes. Since aquaporin expression is modulated by microtubule-dependent trafficking between endoplasmic reticulum and cell membranes, we used the microtubule poison colchicine to assess the importance of aquaporins in mediating GTB. The effects of colchicine 1.6×10−4m on proximal tubule volume reabsorption was tested on 48 nephrons of ten rats by micropuncture techniques. Thirty proximal tubules were sampled from the last proximal convolution before, and recollected during and again after the microinjection (MIJ), into the early proximal convolution or Bowman's space, of colchicine added to a Ringer solution. We studied 18 proximal tubules in the same way before, during and after the microperfusion (MP) of colchicine added to an ultrafiltrate of plasma into the peritubular capillaries. During MIJ, SNGFR did not change significantly from baseline (17.7±1.3 vs 20.9±1.8 nl min−1, P>0.12). Post-control values were superimposable upon their paired pre-MIJ controls, when available (15.8±1.3 vs 13.5±1.5 nl min−1, P>0.25). The measurements of percentage reabsorption (49±5 during baseline, 45±7 during MIJ, and 55±5 in post-control, P>0.6) and absolute reabsorption (8.1±0.7, 11.1±2.2, and 7.9±1.3 nl min−1, respectively, P>0.18) were also unchanged. The three average measurements obtained in control conditions, during MP and again in post-MP control were not significantly different for SNGFR (19.8±3.0, 20.0±4.7, and 20.2±3.5 nl min−1, P>0.48), percentage (55±3, 59±5, and 47±3%, P>0.35) and absolute reabsorptions (12.5±2.2, 12.4±4.6, and 9.4±1.0 nl min−1, respectively, P>0.42). MIJ and MP of vehicles were devoid of any measurable effect. Colchicine does not acutely affect volume reabsorption in the proximal tubule. Aquaporin trafficking, if any, is not involved in mediating glomerulotubular balance in the proximal tubule, although aquaporin expression and function could still be important, although regulated by mechanisms different from microtubule-dependent shuttling between endoplasmic reticulum and BB.

Capillary solid-phase extraction–tandem mass spectrometry for fast quantification of free concentrations of tolterodine and two metabolites in ultrafiltered plasma samples

A capillary solid-phase extraction (SPE) system has been coupled directly to electrospray tandem mass spectrometry for quantification of free tolterodine and metabolite concentrations in plasma. The unbound fraction of these compounds was obtained by ultrafiltration of plasma. The ultrafiltrate was directly injected onto the SPE capillary (4 mm×200 μm, 5 μm C 18). After desalting and clean-up of the sample, the analytes were eluted in backflush mode with methanol–1 m M triethylamine (70:30, v/v), providing considerable solute focusing. Elution from the SPE capillary was improved by inserting a short trapping capillary between the SPE capillary and the MS interface, by which analyte focusing was increased. The unresolved compounds eluted simultaneously with the remaining matrix compounds and were detected in a multiple-reaction monitoring (MRM) mode. No interference of the sample matrix on detection was observed, allowing aqueous standards to be used for calibration. Linear calibration curves were obtained between 0.05 and 1000 ng/ml (corresponding to 150 p M–3 μ M) and the limit of detection was 50 pg/ml injecting 10 μl. Equilibration of the SPE capillary, sample loading, elution and detection took less then 6 min per sample.

Unraveling the mechanisms of glomerular ultrafiltration: nephrin, a key component of the slit diaphragm.

Ultrafiltration of plasma during formation of the primary urine in the glomeruli is a major function of the kidney. The glomerulus is a tuft of anastomosing capillaries located within Bowman's capsule. The glomerular capillary wall has a complex morphology that facilitates a unique filtration

In vivo monitoring of gabapentin in rats: a microdialysis study coupled to capillary electrophoresis and laser-induced fluorescence detection.

Gabapentin (GP) is a new anticonvulsant used in refractory epilepsy. Few studies have monitored the drug in vivo. We report the combination of capillary electrophoresis and laser-induced fluorescence detection (CZE-LIFD) with brain microdialysis and plasma ultrafiltration in an attempt to measure GP and offer an alternative technique for pharmacokinetic studies. We found that CZE-LIFD is capable of linearly measuring 10(-7)-10(-9) M GP in a 1 nL volume. It was also demonstrated that it is possible to monitor GP in prefrontal cortex dialysates and plasma in rats. It is concluded that the method permits in vivo monitoring of the drug in pharmacological as well as in clinical studies.

Influence of assay methodology on the measurement of free serum ceftriaxone concentrations.

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

Glomerular Basement Membrane: IDENTIFICATION OF A NOVEL DISULFIDE-CROSS-LINKED NETWORK OF α3, α4, AND α5 CHAINS OF TYPE IV COLLAGEN AND ITS IMPLICATIONS FOR THE PATHOGENESIS OF ALPORT SYNDROME

Glomerular basement membrane (GBM) plays a crucial function in the ultrafiltration of blood plasma by the kidney. This function is impaired in Alport syndrome, a hereditary disorder that is caused by mutations in the gene encoding type IV collagen, but it is not known how the mutations lead to a defective GBM. In the present study, the supramolecular organization of type IV collagen of GBM was investigated. This was accomplished by using pseudolysin (EC 3.4.24.26) digestion to excise truncated triple-helical protomers for structural studies. Two distinct sets of truncated protomers were solubilized, one at 4 degrees C and the other at 25 degrees C, and their chain composition was determined by use of monoclonal antibodies. The 4 degrees C protomers comprise the alpha1(IV) and alpha2(IV) chains, whereas the 25 degrees C protomers comprised mainly alpha3(IV), alpha4(IV), and alpha5(IV) chains along with some alpha1(IV) and alpha2(IV) chains. The structure of the 25 degrees C protomers was examined by electron microscopy and was found to be characterized by a network containing loops and supercoiled triple helices, which are stabilized by disulfide cross-links between alpha3(IV), alpha4(IV), and alpha5(IV) chains. These results establish a conceptual framework to explain several features of the GBM abnormalities of Alport syndrome. In particular, the alpha3(IV). alpha4(IV).alpha5(IV) network, involving a covalent linkage between these chains, suggests a molecular basis for the conundrum in which mutations in the gene encoding the alpha5(IV) chain cause defective assembly of not only alpha5(IV) chain but also the alpha3(IV) and alpha4(IV) chains in the GBM of patients with Alport syndrome.

Role of NO in cyclosporin nephrotoxicity: effects of chronic NO inhibition and NO synthases gene expression.

The role of nitric oxide (NO) during cyclosporin renal vasoconstriction was evaluated by glomerular hemodynamic and histological changes produced by chronic NO synthesis inhibition and neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) NO syntheses mRNA expression in renal cortex and medulla. Uninephrectomized rats treated during 7 days with vehicle (Veh), cyclosporin A (CsA) 30 mg/kg, CsA + nitro-L-arginine methyl ester (L-NAME), and Veh + L-NAME (10 mg/dl) in the drinking water were studied. Increase in arterial pressure and afferent and efferent resistances, as well as decrease in glomerular plasma flow, ultrafiltration coefficient, and single-nephron glomerular filtration rate were significantly greater with CsA + L-NAME than with CsA alone. The increase in afferent resistance was higher with CsA + L-NAME than with Veh + L-NAME. In addition, glomerular thrombosis, proximal tubular vacuolization, and arteriolar thickening were more prominent. In renal cortex, eNOS mRNA expression exhibited a 2.7-fold increase in CsA, whereas, in medulla, nNOS and iNOs expression were lower in CsA than in Veh, while eNOS tended to increase. Our results support the hypothesis that NO synthesis is enhanced at cortical level during CsA nephrotoxicity, counterbalancing predominantly preglomerular vasoconstriction. Higher NO production could be the result of increased eNOS mRNA expression.

Bicarbonate promotes a cleavage of lactone ring of dehydroascorbate

The half-life of dehydroascorbate (DHA) in human plasma is only a few minutes. This DHA disappearance is caused by a cleavage of lactone ring. Similarly, when DHA was incubated in Dulbecco's modified Eagle's medium (D-MEM), which stood in atmosphere of 5% CO 2–95% air, the rapid transformation of DHA into 2,3-diketogulonate (2,3-DKG) is also observed. These observations suggest that both human plasma and D-MEM contain a common component, which promotes the hydrolysis of DHA. In the present study, this component was identified to be bicarbonate which acts as a general base catalyst. Direct evidence for this mechanism was obtained as follows: (1) significant hydrolysis of DHA in the bicarbonate-free D-MEM (pH 7.40) was not observed; (2) hydrolysis of DHA in Tris–HCl buffer at constant pH (7.4) increases with increasing bicarbonate concentration; and (3) significant hydrolysis of DHA in the decarbonated ultrafiltrate of plasma was not observed. These results suggest that DHA hydrolysis may be controlled by the variation of CO 2 pressure in circulating blood.

Diagnostic Interpretation of Pericardial Fluids

Pericardial fluid, normally measuring 15–35 mL, is mainly an ultrafiltrate of plasma, possibly with some overflow of myocardial interstitial fluid and lymph drainage.1 Protein concentration is lower in pericardial fluid than in plasma but albumin is relatively increased, owing to its lower molecular weight and ease of transmembrane transport. Electrolyte concentrations are as predicted for a plasma filtrate, yielding fluid osmolarity consistent with such an ultrafiltrate and therefore less than plasma osmolarity.

Determination of amikacin in human plasma by high-performance capillary electrophoresis with fluorescence detection.

A selective and reproducible high-performance capillary electrophoretic (HPCE) method for the quantification of amikacin (AMK), an aminocyclitol antibiotic, in human plasma, has been developed for use in clinical laboratory tests. The method involves ultrafiltration (UF) of plasma before derivatization with the fluorescence derivatization reagent 1-methoxy-carbonylindolizine-3, 5-dicarbaldehyde at room temperature for 15 min in the dark. An aliquot of the derivatives is directly introduced into the fused-silica capillary [75 cm (effective length) x 50 microns I.D.] at the anode side by dynamic compression injection (50 hPa for 6 s). After electrophoresis with 40 mM SDS-20 mM phosphate-borate buffer (pH 7) in the micellar electrokinetic chromatography (MEKC) mode at 30 kV, the derivative had a retention time of 16.7 min and was detected by fluorescence intensity at 482 nm (with irradiation at 414 nm). The precision (n = 5) of the method is 4.08 and 1.59% (C.V.) at the 50 and 100 micrograms AMK/ml plasma levels, respectively. Linearity (r = 0.998) was established over the concentration range 5-100 mg of AMK/ml plasma and the detection limit (at a signal-to-noise ratio of 3) is 0.5 microgram AMK/ml plasma. This assay method could potentially have wider application in the determination of other aminocyclitol antibiotics, such as arbekacin, dibekacin, kanamycin, in human plasma as well as of AMK.

Determination of unbound etoposide concentration in ultrafiltered plasma by high-performance liquid chromatography with fluorimetric detection.

Etoposide is a highly protein bound drug, and monitoring the concentration of free drug could help individualize dosage in oncological patients. The cost and difficulty of the standard techniques (equilibration dialysis) has hampered the monitoring of free drugs. We describe a simple HPLC method for the measurement of free etoposide concentration in plasma. Sample preparation involves the ultrafiltration of plasma by a Centrifree device for 30 min at 2000 g and extraction with chloroform. The isocratic separation is performed with a mu Bondapak phenyl analytical column. Fluorimetric detection is used (288-328 nm excitation and emission wavelengths). Linearity of the calibration curve is excellent between 0.05 and 1 microgram/ml. Accuracy and precision are reported at the concentrations 0.06 and 0.4 microgram/ml: within-run accuracy is 10% and 6.2%, respectively; between-run accuracy is < or = 1%; within-run coefficients of variation (C.V.) are 10.6 and 5.0%; between-run C.V. are 11.6 and 6.8% respectively. The range of the assay is 0.05 to 1 microgram/ml. The feasibility of the technique has been tested in 7 patients treated with oral etoposide for hepatocarcinoma (mean protein binding 91%). We found no interference from endogenous substances, co-administered drugs (alizapride, furosemide, ranitidine) and other antineoplastic agents (doxorubicine, idarubicine, vinblastine, vinorelbine).

A high resolution PIXE measurement for blood plasma ultrafiltrate. Application to loosely bound copper.

A new technique for investigation of elemental concentrations in subfractions of blood plasma is presented. The method is composed of the ultrafiltration of plasma in the presence of ethylenediaminetetraacetic acid (EDTA) and the measurement of the elemental composition by proton-induced X-ray emission (PIXE). The blood samples were collected from both healthy persons and patients suffering from breast cancer. The main emphasis in this study was on the determination of loosely bound copper (Cu) in plasma subfractions containing substances with molecular mass under 10,000, but zinc (Zn) and iron (Fe) contents of these fractions were also determined. The detection limits obtained with this method for Cu, An and Fe were approximately 10 ppb (wet wt).

Spectrophotometric determination of the cardioprotective drug GP531 in human plasma

A simple spectrophotometric assay has been developed for determining concentrations of the new cardioprotective agent, GP531, in human plasma. The method is adapted from the Bratton-Marshall assay (BMA) for the detection of primary aromatic amines. The assay can be used to measure plasma concentrations of the drug during i.v. infusion administration to patients. The limit of quantitation of the assay is 1 μg/ml using a 0.8 ml sample ultrafiltrate of plasma. The colorimetric assay correlates well with a previously described high performance liquid chromatographic (HPLC) procedure.

Pharmacokinetics of thiopental and pentobarbital enantiomers after intravenous administration of racemic thiopental.

We studied the pharmacokinetics of thiopental enantiomers in 14 healthy patients aged 37-73 yr receiving racemic thiopental by intravenous (IV) bolus or IV infusion. Plasma concentration of each enantiomer was measured by chiral high-performance liquid chromatography. After IV bolus, the total plasma clearance (CL) (295 +/- 132 mL/min) and volume of distribution at steady state (Vss) (139 +/- 38.5 L) of R-thiopental were significantly greater than those of S-thiopental (230 +/- 104 mL/min and 114 +/- 47.5 L, respectively). The plasma unbound fraction (fu) was determined by ultrafiltration of plasma from six healthy volunteers. The fu of R-thiopental (12.4% +/- 0.6%) was significantly greater than that of S-thiopental (10.0% +/- 1.0%). When the CL and Vss of the two enantiomers were corrected for the difference in mean fu, there were no significant differences between enantiomers for these variables. As the 20%-30% difference between the enantiomers in total CL and total Vss could be accounted for by stereoselectivity in fu, these differences are not likely to be clinically significant. During 105-180 min IV infusion of racemic thiopental to the other patients, there was no difference between enantiomers in mean plasma concentrations of total or unbound thiopental or total pentobarbital, a major metabolite of thiopental (P > 0.05). Therefore, it is appropriate to relate pharmacodynamic effects to racemic plasma concentrations of thiopental during IV infusion of racemic thiopental.

Pharmacokinetics of Thiopental and Pentobarbital Enantiomers After Intravenous Administration of Racemic Thiopental

We studied the pharmacokinetics of thiopental enantiomers in 14 healthy patients aged 37-73 yr receiving racemic thiopental by intravenous (IV) bolus or IV infusion. Plasma concentration of each enantiomer was measured by chiral high-performance liquid chromatography. After IV bolus, the total plasma clearance (CL) (295 +/- 132 mL/min) and volume of distribution at steady state (Vss) (139 +/- 38.5 L) of R-thiopental were significantly greater than those of S-thiopental (230 +/- 104 mL/min and 114 +/- 47.5 L, respectively). The plasma unbound fraction (fu) was determined by ultrafiltration of plasma from six healthy volunteers. The fu of R-thiopental (12.4% +/- 0.6%) was significantly greater than that of S-thiopental (10.0% +/- 1.0%). When the CL and Vss of the two enantiomers were corrected for the difference in mean fu, there were no significant differences between enantiomers for these variables. As the 20%-30% difference between the enantiomers in total CL and total Vss could be accounted for by stereoselectivity in fu, these differences are not likely to be clinically significant. During 105-180 min IV infusion of racemic thiopental to the other patients, there was no difference between enantiomers in mean plasma concentrations of total or unbound thiopental or total pentobarbital, a major metabolite of thiopental (P > 0.05). Therefore, it is appropriate to relate pharmacodynamic effects to racemic plasma concentrations of thiopental during IV infusion of racemic thiopental. (Anesth Analg 1996;83:552-8)

Determination of platinum in protein-bound CDDP and DBP by inductively coupled plasma optical emission spectrometry and electrothermal atomic absorption spectrometry

The plasma protein binding ability of cis-diammine-{[bis(phosphonatomethyl)amino]acetato(2–)-O1, N1}platinum(II)(DBP), a tumour-inhibiting platinum phosphonato compound with osteotropic properties, was compared with that of the well established anticancer drug cisplatin (CDDP). The separation of the plasma protein-platinum adducts was performed by a fractionated ultrafiltration experiment. The platinum levels in the protein fraction and the ultrafiltrate of the blood plasma were detected with an échelle-based ICP-OES system with an ultrasonic nebulizer (USN) and by ETAAS with Zeeman-effect background correction. The over-all performance of ETAAS was better than that of ICP-OES. Owing to strong, protein-induced matrix effects in the USN, the ICP-OES measurements resulted in lower platinum levels in the protein fraction of blood. In the determination of platinum in the ultrafiltrate, both systems showed good performance. In comparison with CDDP, DBP shows a significant tendency to bind to plasma proteins of lower molecular mass.