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Internal membranes and laminates for adaptation of amperometric enzyme electrodes to direct biofluid analysis.

This study describes the use of discrete, highly selective covering inner membranes in amperometric enzyme electrodes for direct biofluid analysis. Such inner membranes when interposed between the enzyme layer and the electrode working surface has been found to be both selective, as well as biocompatible. Cellulose acetate or detergent (Triton X-100 and Tween 80) modified cellulose acetate and polyethersulphone polymeric membranes, were both found to be highly selective against electroactive solutes normally present in biofluids. The inclusion of such inner perm-selective membranes greatly enhanced the biocompatibility of the enzyme electrode and reduced the passivation of the working electrode on exposure to whole blood or serum. The study indicates that electrode drift in biological solutions may also be due to passivation of the working electrode by small diffusible surface active species.

Biocompatible Materials↗

Multicomponent investigations of the hydrogen peroxide- and hydroxyl radical-scavenging antioxidant capacities of biofluids: the roles of endogenous pyruvate and lactate. Relevance to inflammatory joint diseases.

High field proton (1H) nuclear magnetic resonance (NMR) analysis of biofluids (health human blood sera and inflammatory knee-joint synovial fluids) has been employed to evaluate the hydrogen peroxide (H2O2)- and hydroxyl radical (0OH)- scavenging antioxidant capacities of a range of polar, low-molecular-mass endogenous metabolites therein. Data obtained indicate that consumption of H2O2 by pyruvate (generating acetate and CO2 via an oxidative decarboxylation reaction) and 0OH radical by lactate (generating pyruvate, and subsequently acetate and CO2) may serve to protect alternative biofluid components (e.g., macromolecules) against reactive oxygen species-mediated oxidative damage in vivo. The mechanistic, physiological and potential therapeutic implications of these results are discussed with special reference to inflammatory joint diseases.

Adult↗

Analysis of biofluids for flunitrazepam and metabolites by electrospray liquid chromatography/mass spectrometry.

A rapid and sensitive liquid chromatography/electrospray ionization mass spectrometry (LC/MS) procedure has been developed for the analysis of biofluids containing flunitrazepam and its metabolites. Specimens were spiked with deuterated analogs of the analytes. Urine specimens were enzymatically hydrolyzed and blood specimens were untreated. Extractions were carried out using CleanScreen DAU SPE cartridges. The drugs were separated on a C18 column using a methanol-water-ammonium hydroxide (60:40: 0.03 v/v) mobile phase. After determination of base peaks using full scan mass spectrometry, the mass spectrometry method was optimized to operate in selected-ion monitoring (SIM) mode for the base peak of each analyte. Positive findings were confirmed by LC/MS/MS using the same mobile phase and column. This analytical procedure allows for the detection of low levels of flunitrazepam and metabolites in biofluids. It is useful for ascertaining the role of flunitrazepam in cases of drug-facilitated sexual assault.

Anti-Anxiety Agents↗

Analysis of biofluids and chemical mixtures in non-deuterated solvents with 1H diffusion-weighted PFG phase-sensitive double-quantum NMR spectroscopy.

Two different types of one- and two-dimensional diffusion-weighted H phase-sensitive pulsed field gradient double-quantum experiments (DWDQ) are presented and their application to a biological plasma sample is shown. The remarkable solvent suppression achieved with these pulse sequences and the high sensitivity of these experiments allows the determination of the diffusion coefficients of molecules at low concentration dissolved in non-deuterated solvents. Biofluids and complex chemical mixtures from combinatorial chemistry and from natural sources can be analysed with these experiments, and the different molecular species of the mixtures can be identified and assigned, based on their different diffusion coefficients.

Amino Acids↗

In vivo biofluid dynamic imaging in the developing zebrafish.

Flow-structure interactions are ubiquitous in nature, and are important factors in the proper development of form and function in living organisms. In order to uncover the mechanisms by which flow-structure interactions affect vertebrate development, we first need to establish the techniques necessary to quantitatively describe the fluid flow environment within the embryo. To do this, we must bring dynamic, in vivo imaging methods to bear on living systems. Traditional avian and mammalian model systems can be problematic in this regard. The zebrafish (Danio rerio) is widely accepted as an excellent model organism for the study of vertebrate biology, as it shows substantial anatomical and genetic conservation with higher vertebrates, including humans. Their small size, optical transparency, and external development make zebrafish the ideal model system for dynamic imaging. This article reviews the current state of research in imaging biofluid flow within and around developing zebrafish embryos, with an emphasis on dynamic imaging modalities.

Animals↗

Human bronchoalveolar lavage: biofluid analysis with special emphasis on sample preparation.

Respiratory diseases are an important health problem throughout the world. Whether caused by industrial pollutants, infections, smoking, cancer or metabolic diseases, damage to the lungs and airways often lead to morbidity or death. Bronchoalveolar lavage (BAL) obtained by fiber-optic bronchoscopy is a biofluid mirroring the expression of normally secreted pulmonary proteins and the products of activated cells and destructive processes. The characterization of the proteome within this compartment provides an opportunity to establish temporal and prognostic indicators of airway disease. The objective of this study was to develop methods of analysis of BAL samples, which achieved the highest level of annotation of the expression map of this proteome. We have optimized the process of sample preparation after investigating a variety of techniques including dialysis, ultramembrane filtration, precipitation and gel filtration. We have further studied methods to remove albumin from BAL in order to unmask proteins hidden on two-dimensional gels. In a pilot application of the method, BAL protein profiles obtained from healthy nonsmokers and smokers at risk for developing chronic obstructive pulmonary disease showed distinct differences.

Albumins↗

Determining 15N to 14N ratios in biofluids by single-pulse 1H nuclear magnetic resonance.

In the 1H NMR spectra of acidified biofluids from our studies of nitrogen metabolism the 1:1 doublet signal (1J-1H-15N = 73.2 Hz) from the protons of 15N-enriched ammonia is clearly resolved from the 1:1:1 triplet signal (1J-1H-14N = 52.3 Hz) from (naturally abundant) 14N ammonia. The five spectral lines, infinitely broad at neutral pH, produce a "pseudo-pentet" at 7.1 ppm from which the fractional 15N-enrichment is easily calculated. 1H NMR spectra of plasma and urine from rats infused with 15N-enriched ammonium acetate illustrate the technique. The greater sensitivity of 1H NMR over 15N and 14N NMR spectroscopy suggests 15N-ammonia determinations by this method will compare favorably with other methods for determining nitrogen labeling.

Animals↗

1H NMR investigations of the molecular nature of low-molecular-mass calcium ions in biofluids.

High-resolution 1H NMR spectroscopy was employed to explore the complexation of Ca2+ by low-molecular-mass biomolecules in human saliva. The results acquired revealed that the organic acid anion (OAA) citrate acts as a powerful oxygen-donor chelator for salivary Ca2+, and accurate determination of its resonances' frequencies and spin-system pattern could be successfully utilized to determine its degree of saturation with this metal ion. Computer modelling studies demonstrated that the OAA lactate is the only competing salivary Ca2+ complexant available. Moreover, the Ca2+-complexation status of salivary citrate is substantially modified by dentifrice-mediated elevations in its concentration. 1H NMR analysis was also applied to determinations of the Ca2+ saturation status of citrate in a variety of alternative biofluids and the biochemical significance of these results is discussed.

Amniotic Fluid↗

Capillary electrophoretic analyses of drugs in body fluids: sample pretreatment and methods for direct injection of biofluids.

A variety of strategies for the analysis of biological samples by capillary electrophoresis (CE) are described, with particular emphasis on the determination of drugs and metabolites. Analytical methods involving extensive sample pretreatment before CE analysis are considered, as well as strategies for directly injecting untreated biofluids. The application in CE of techniques common in liquid chromatography is first described, e.g. protein precipitation, liquid-liquid extraction and solid-phase extraction. On-capillary methods of sample concentration are considered. Approaches to performing CE assays of urine and plasma, without prior sample treatment, are described. The use of both capillary zone electrophoresis and micellar electrokinetic chromatography for direct-injection assays is compared for both urine and plasma analyses, and capillary washing strategies are discussed. Finally, direct-injection microanalyses are mentioned.

Body Fluids↗

Application of the one-dimensional TOCSY pulse sequence in 750 MHz 1H-NMR spectroscopy for assignment of endogenous metabolite resonances in biofluids.

The complex 1H-NMR spectrum arising from an intact biofluid has been simplified using a one-dimensional homonuclear polarization transfer experiment (known as TOCSY or HOHAHA). This approach establishes connectivity between sequentially coupled multiplets, and the method is illustrated by the confirmation of the chemical shifts and hence resonance assignment of a number of endogenous metabolites in the 750 MHz 1H-NMR spectrum of seminal fluid. This has allowed the detection and assignment of pyroglutamate and uracil in this fluid for the first time.

Computer Simulation↗

Determination of peplomycin in mouse tissues and biofluids by radioimmunoassay.

Peplomycin, an antitumour antibiotic analogue of bleomycin, was measured in mouse tissues using a rapid radioimmunoassay. Antiserum, obtained by immunizing rabbits with peplomycin-bovine serum albumin conjugate, showed no significant cross-reactivity with the closely related peplomycin analogues bleomycin and liblomycin, nor with a number of other structurally unrelated antitumour drugs. The assay is sensitive and can detect peplomycin levels as low as 2 ng ml-1. The relative intra- and inter-assay standard deviation is < or = 5%, indicating good assay reproducibility. Peplomycin levels in mouse tissues were easily determined without extraction. Fifteen minutes after administration of a single intraperitoneal dose of peplomycin at 8.5 mg kg-1 (1/10 of LD50), high drug levels were found in plasma (46 micrograms ml-1), kidneys (38 micrograms g-1), urine and bladder (32 micrograms ml-1), followed by gastrointestinal tract (13 micrograms g-1), lung (8 micrograms g-1), spleen (3.7 micrograms g-1), heart (3.6 micrograms g-1), gall bladder (2.7 micrograms g-1), liver (2 micrograms g-1), and brain (0.6 microgram g-1). The total amount of drug in all these organs accounted for more than 80% of the dose administered. We conclude that the radioimmunoassay is sensitive and reproducible and is an ideal tool for measuring peplomycin in tissues and biofluids for pharmacological studies.

Animals↗

Capillary electrophoresis with electrochemiluminescence detection for measurement of aspartate aminotransferase and alanine aminotransferase activities in biofluids.

A new sensitive assay for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in biofluids was developed, based on the separation and detection of alanine, glutamate, and aspartate using capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection. The three amino acids were separated in 5 mM phosphate of pH 2.1 as background electrolyte, and detected on a 500 microm platinum disk electrode at 1.2V (versus Ag/AgCl) in the presence of 10 mM tris(2,2'-bipyridyl)ruthenium(II) dissolved in 80 mM phosphate of pH 10.5. A mass detection limit of 37.3 fmol (or 81.5 fmol) for glutamate, corresponding to the product in the enzyme reaction catalyzed by 1.24 x 10(-9)U AST (or 2.72 x 10(-9)U ALT) in a 30 min reaction period, was achieved. This assay was applied to investigate the cytotoxicity effect of ethanol on HepG2 cells and differentiating nonalcoholic steatohepatitis (NASH) from alcoholic liver disease, indicating that the technique is promising for the application in the cell biological and clinical fields.

Alanine↗

An introduction to biofluid mechanics--basic models and applications.

Cardiovascular disease is the primary cause of morbidity and mortality in the western world. Complex hemodynamics play a critical role in the development of atherosclerosis and the processes of aging, as well as many other disease processes. Biofluid mechanics play a major role in the cardiovascular system and it is important to understand the forces and movement of blood cells and whole blood as well as the interaction between blood cells and the vessel wall. Fundamental fluid mechanical, which are important for the understanding of the blood flow in the cardiovascular circulatory system of the human body aspects are presented. Measurement techniques for model studies such as LDA, ultrasound, and MRI studies will be discussed. Viscosity and flow behavior changes specifically the creation of vortices and flow disturbances can be used to show how medication can influence flow behavior. Experiments have shown that hemodynamics may have a strong influence on the creation of aneurysms and varicose veins. Other factors such as vessel wall structure are also important. In preliminary studies, it has been demonstrated that geometry and elasticity of vessel walls help determine flow behavior. High velocity fluctuations indicate flow disturbances that should be avoided. Health care practitioners must understand fluid dynamic factors such as flow rate ratio, pressure and velocity gradients, and flow behavior, velocity distribution, shear stress on the wall and on blood cells. These mechanical factors are largely responsible for the deposit of blood cells and lipids, a leading cause of atherosclerosis. The interaction between blood cells and of the cells with the vessel, leads to the formation of plaques and agglomerations. These deposits are found predominantly at arterial bends and bifurcations where blood flow is disturbed, where a secondary flow is created, and where flow separation regions are found. Experiments on hemodynamic effects in elastic silicon rubber models of the cardiovascular system with flow wire, stents, or patches for vessel surgery will be discussed. These studies can be important in improving diagnostics and therapeutic applications.

Animals↗

Modeling of spontaneous penetration of viscoelastic fluids and biofluids into capillaries.

A theoretical model was developed to describe the dynamics of spontaneous penetration of viscoelastic fluids into capillaries. The model agrees quantitatively with recent experiments on absorption of droplets of polymer solutions by glass capillaries [A.V. Bazilevsky, K.G. Kornev, A.N. Rozhkov, A.V. Neimark, J. Colloid Interface Sci. (2003)]. The rate of penetration progressively reduces with the increase in fluid elasticity. Analysis revealed two main contributions to the viscoelastic drag of the liquid column: (i) viscous resistance, which is independent of fluid elasticity, and (ii) viscoelastic resistance, known as the Weissenberg effect. We analytically derived an augmented Bosanquet equation for the maximal velocity of penetration by balancing capillary, inertia, and viscoelastic forces. For slow creep of a liquid column, the Lucas-Washburn equation was modified by accounting for the Weissenberg effect. A series of numerical calculations were performed to demonstrate characteristic features of absorption of fluids at different conditions. This article also discusses some problems specific to absorption of biofluids. We show that deformations of cell membranes in the external converging flow may cause their rupture at the pore entrance.

Data Interpretation, Statistical↗

The estimation of the bioaccessibility of heavy metals in soils using artificial biofluids by two novel methods: mass-balance and soil recapture.

The possible human health effects resulting from the ingestion of soil bound heavy metals can be poorly estimated if concentration of total metals in soil, rather than bioavailable fraction of metals, are incorporated into dose calculations. Information regarding bioavailability often is obtained from animal studies, which are not easily conducted and still may not represent human conditions. A rapid simulation of the bioaccessible fraction of contaminant in a soil, in which that fraction is mass soluble in gastrointestinal tract fluids, has been employed in an in vitro sequential extraction technique. Using a mass-balance analytical approach to measure bioaccessibility in four soils, the results indicated that each metal had a bioaccessible fraction less than its total metal content. Lead (Pb) in Standard Reference Material, Montana SRM 2710, was found to be 62 +/- 1% bioaccessible; Pb in contaminated soil collected from Bunker Hill, ID, USA was 70 +/- 11%. Lead in Jersey City, NJ, USA slag material was only 39 +/- 14% bioaccessible while Pb in a residential soil was 69%. Arsenic (As) and chromium (Cr) data from select soils also have bioaccessibility less than the corresponding total metal in soil, with 41 +/- 2% As in a residential soil, 66 +/- 8% As in SRM 2710, and 34 +/- 14% Cr in Jersey City slag material. Recovering the soil at the end of the in vitro extraction allowed for the determination of the insoluble fraction of total metal in soil. This recaptured soil metal mass was a valuable measurement since it greatly reduced analysis and therefore labor and time, yet also provided a reasonable estimate of bioaccessibility. It also allowed for calculation of a bioaccessibility value in a soil containing very low metal mass, which would otherwise have resulted in a non-detectable concentration at the dilutions required in the synthetic human biofluid system.

Biological Availability↗

Reaction of the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate with human biofluids.

Using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS), an oxidant was previously detected in the plasma of patients with renal failure and the synovial tissue of rheumatoid arthritis patients. This oxidant has been shown to react with DBNBS to give a 3-line electron paramagnetic resonance (EPR) spectrum, previously assigned to the DBNBS radical cation (DBNBS*(+). However, confusion has arisen as to whether this paramagnetic species is indeed DBNBS*(+) or, rather, the DBNBS sulfite radical adduct (DBNBS-SO(3)*(-)). In the present study, DBNBS*(+) (a(N)=1.32 mT) was distinguished from DBNBS-SO(3)*(-) (a(N)=1.32 mT with an additional splitting of a(H)=0.06 mT) by (a) using different EPR parameters, (b) determining the effect of addition of sulfite on the EPR spectrum resulting from the incubation of DBNBS with either human biofluids or the horseradish peroxidase (HRP)-hydrogen peroxide (H(2)O(2)) system, and (c) replacing DBNBS with its analogues (DBNBS-d(2,) DBNBS-15N and DBNBS-d(2)-15N) in the two systems.

Benzenesulfonates↗

Accurate titration of avidin and streptavidin with biotin-fluorophore conjugates in complex, colored biofluids.

A new fluorimetric assay is presented for the specific and reliable quantitation of >/=2 nM avidin and streptavidin. The assay is based on pronounced changes in the fluorescence properties of commercial fluorescein-biotin, or of a newly synthesized biotin-poly(ethylene glycol)-pyrene conjugate, which occur upon binding to avidin and streptavidin. Accurate measurement of (strept)avidin in complex, colored biofluids, such as crude egg white or serum relies on a simple titration protocol. Only occasional recalibration of the reagent solution is required. Due to these merits the proposed assay is particularly suited for rapid measurement of few samples on short notice, for functional control of (strept)avidin-containing reagents after storage, and for the monitoring of (strept)avidin concentrations in large scale processes.

Animals↗

Validation of a highly sensitive ICP-MS method for the determination of platinum in biofluids: application to clinical pharmacokinetic studies with oxaliplatin.

ELOXATIN (Oxaliplatin) is a novel platinum containing anti-cancer agent with a diaminocyclohexane carrier ligand which has been approved in several major European countries. Clinical studies have demonstrated that the compound exhibits marked activity against colorectal cancers in combination with 5-fluorouracil (5-FU). The aim of this work was to develop and validate a highly sensitive inductively coupled plasma mass spectrometry assay for the determination of oxaliplatin-derived platinum in plasma ultrafiltrate, plasma and whole blood and to apply this technique to clinical pharmacokinetic studies with oxaliplatin. Ultratrace detection of platinum in plasma ultrafiltrate was achieved using ultrasonic nebulisation combined with ICP-MS. This technique allows detection of platinum at the 0.001 microg Pt/ml level in only 100 microl of matrix. Assays in blood and plasma utilised a standard Meinhardt nebuliser and spray chamber, achieving detection limits of 0.1 microg Pt/ml in 100 and 200 microl of matrix, respectively. The assays were validated (accuracy and precision within +/- 15%) over the concentration ranges: 0.001-0.250 microg Pt/ml in plasma ultrafiltrate and 0.1-10 microg Pt/ml for plasma and whole blood. The effect of sample digestion. dilution, long term frozen storage and quantitation in the presence of 5-FU were also investigated and validated. The method was used to monitor platinum exposure following oxaliplatin administration (130 mg/m2) to cancer patients. Following a 2 h i.v. infusion, peak platinum levels declined in a triphasic manner in all blood compartments. Free platinum was detected in plasma ultrafiltrate at low levels (0.001 0.010 microg Pt/ml) for up to 3 weeks. In conclusion, a highly sensitive and specific assay has been developed for the determination of platinum in biofluids. This method enabled characterisation of the long term exposure to platinum in patients following oxaliplatin treatment.

Antineoplastic Agents↗