Analytical determination of orthophosphate in water

Methods for orthophosphate determination and the problems of interferences are reviewed.An important group of methods utilizes the phosphomolybdate complex. The complexation step, the reduction step and the extraction step are treated separately and alternative procedures compared.Another group of methods uses ion association complexes; they are primarily used in physiology and not commonly used in water analyses today.Enzymatic methods for orthophosphate analysis in natural waters have been developed lately and are ready for application in selected waterbodies.Flame spectroscopic, fluorometric, gas chromatographic, ion exclusion chromatographic, inductively coupled plasma and other methods are also shortly presented.Radiobiological bioassays for orthophosphate are also available.In conclusion it was emphasized that the most common and reliable technique still is the molybdenum blue method as modified by Murphy & Riley (1962).The need for more specific and sensitive methods is particularly strong in investigations of phosphorus turnover and phosphorus limitation in natural waters. For these purposes the enzymatic phosphatase methods has advantages due to their specificity for orthophosphate and they might offer an alternative to the molybdenum blue method.     

High-throughput three-dimensional protein structure determination

In the wake of finished genomic sequencing projects, high-throughput analysis techniques are being developed in various fields of functional genomics. Of special interest in this regard is the three-dimensional structure analysis of proteins by X-ray crystallography and NMR spectroscopy, which has been characterized by distinctly low-throughput in the past. A number of recent advances in instrumentation and software are promising to radically change this situation, leaving the production of suitable protein samples as the sole rate-limiting step in structural analyses.     

The determination of the three-dimensional structure of barley serine proteinase inhibitor 2 by nuclear magnetic resonance, distance geometry and restrained molecular dynamics

The solution structure of the 64 residue structured domain (residues 20-83) of barley serine proteinase inhibitor 2 (BSPI-2) is determined on the basis of 403 interproton distance, 34 phi backbone torsion angle and 26 hydrogen bonding restraints derived from n.m.r. measurements. A total of 11 converged structures were computed using a metric matrix distance geometry algorithm and refined by restrained molecular dynamics. The average rms difference between the final 11 structures and the mean structure obtained by averaging their coordinates is 1.4 +/- 0.2 A for the backbone atoms and 2.1 +/- 0.1 A for all atoms. The overall structure, which is almost identical to that found by X-ray crystallography, is disc shaped and consists of a central four component mixed parallel and antiparallel beta-sheet flanked by a 13 residue alpha-helix on one side and the reactive site loop on the other.     

Analytical derivatization-a tool for determination of orotic acid

Derivatization of orotic acid (OA) into various forms (trimethylsilylderivate, alkyl ester and per-methylated derivate) and their evaluation by GC/MS is described. The tested approach includes ion-exchange SPE clean-up, evaporation and chemical reaction with different types of derivatization agents (N,O-bis-(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane, butanol with acetylchloride and ethereal solution of diazomethane). Derivate originated in the reaction with diazomethane was used for determination of urinary orotic acid by GC/MS. Detection limit of 0.28 micromol l(-1) was reached using the ion 82 m/z in single ion monitoring (SIM) mode. Linearity of the method was tested within the range of 3.4-2503.4 micromol l(-1) covering physiological and pathological levels of orotic acid in urine sample. Recoveries were within the range 93.7-110.6%. Application of the method on the patient with defect of ornithine transcarbamylase (OTC) was demonstrated as well.     

A three-dimensional variable geometry countercurrent model for whole limb heat transfer.

A new formulation of the combined macro and microvascular model for heat transfer in a human arm developed in Song et al. [1] is proposed using a recently developed approximate theory for the heat exchange between countercurrent vessels embedded in a tissue cylinder with surface convection [2]. The latter theory is generalized herein to treat an arm with an arbitrary variation in cross-sectional area and continuous bleed off from the axial vessels to the muscle and cutaneous tissue. The local microvascular temperature field is described by a "hybrid" model which applies the Weinbaum-Jiji [3] and Pennes [4] equations in the peripheral and deeper tissue layers, respectively. To obtain reliable end conditions at the wrist and other model input parameters, a plethysmograph-calorimeter has been used to measure the blood flow distribution between the arm and hand circulations, and hand heat loss. The predictions of the model show good agreement with measurements for the axial surface temperature distribution in the arm and confirm the minimum in the axial temperature variation first observed by Pennes [4] for an arm in a warm environment.     

A stereophotogrammetric method for measurements of ligament structure

A stereophotogrammetric method is presented to reconstruct the course of a curve in the three-dimensional space. This method is exclusively suitable as a non-destructive tool to determine the surface fiberstructure of ligaments, tendons and other organised collagenous structures. In addition, it is a convenient tool to measure the geometry of articular surfaces and other complicated surface shapes.     

Concentrations of trace elements in osteoarthritic knee-joint effusions

Concentrations of the 18 elements, barium (Ba), beryllium (Be), bismuth (Bi), calcium (Ca), cadmium (Cd), cesium (Cs), copper (Cu), lanthanum (La), lithium (Li), magnesium (Mg), molybdenum (Mo), lead (Pb), rubidium (Rb), antimony (Sb), tin (Sn), strontium (Sr), thallium (Tl), and zinc (Zn), were determined in the synovial fluids of osteoarthritic knee joints and in the corresponding sera of 16 patients by inductively coupled plasma-mass spectrometry. Knee-joint effusions have lower elemental concentrations than their corresponding sera. For the essential elements Ca, Cu, Mg, and Zn and for the nonessential and toxic elements Ba, Be, Bi, La, and Sb, this difference was highly significant. Strong positive correlations between concentrations in effusions and sera for the essential elements Cu and Mg and for the nonessential elements Cs, Li, Rb, and Sr could be established. The grade of localized hyperperfusion of the knee region in the blood pool phase of ^99mTc HDP bone scan indicating inflammation did not correlate with any elemental concentration determined. Deceased.     

Cell growth patterns and lens geometry: a quantitative study from three-dimensional reconstructions

The growth of the lens of the sea lamprey, Petromyzon marinus, was studied over the 5 years of larval development. Whole lenses (25) and Golgi-impregnated cells (393) were reconstructed with computer-assisted microscopy. Several cellular geometric parameters (length, width, curvature, surface, volume, shape) were correlated with the position of the cell's base on the lens capsular perimeter. Based on these correlations, the cells formed four groups that correspond to the central anterior, germinative, transitional and cortical fiber zones. A fifth zone, containing nuclear fiber cells, never stained. Lens growth is exponential during the 5 years. The anterior epithelium increases in size and in cell number by cell growth and division. The posterior mass increases in cell number by recruitment and increases in size by cell growth. A model is proposed to account for the size and shape of the lens based upon the coupling of anterior and posterior growth patterns. Four zonal boundaries are defined by changes in cell growth patterns. With growth, cells are subsumed into adjacent zones and zonal boundaries move away from the lens center. We find no support for the suggestion that cells migrate centrally.     

Opioid receptor three-dimensional structures from distance geometry calculations with hydrogen bonding constraints.

Three-dimensional structures of the transmembrane, seven alpha-helical domains and extracellular loops of delta, mu, and kappa opioid receptors, were calculated using the distance geometry algorithm, with hydrogen bonding constraints based on the previously developed general model of the transmembrane alpha-bundle for rhodopsin-like G-protein coupled receptors (Biophys. J. 1997. 70:1963). Each calculated opioid receptor structure has an extensive network of interhelical hydrogen bonds and a ligand-binding crevice that is partially covered by a beta-hairpin formed by the second extracellular loop. The binding cavities consist of an inner "conserved region" composed of 18 residues that are identical in delta, mu, and kappa opioid receptors, and a peripheral "variable region," composed of 19 residues that are different in delta, mu, and kappa subtypes and are responsible for the subtype specificity of various ligands. Sixteen delta-, mu-, or kappa-selective, conformationally constrained peptide and nonpeptide opioid agonists and antagonists and affinity labels were fit into the binding pockets of the opioid receptors. All ligands considered have a similar spatial arrangement in the receptors, with the tyramine moiety of alkaloids or Tyr1 of opioid peptides interacting with conserved residues in the bottom of the pocket and the tyramine N+ and OH groups forming ionic interactions or H-bonds with a conserved aspartate from helix III and a conserved histidine from helix VI, respectively. The central, conformationally constrained fragments of the opioids (the disulfide-bridged cycles of the peptides and various ring structures in the nonpeptide ligands) are oriented approximately perpendicular to the tyramine and directed toward the extracellular surface. The results obtained are qualitatively consistent with ligand affinities, cross-linking studies, and mutagenesis data.     

A three-dimensional model of the rat hindlimb: musculoskeletal geometry and muscle moment arms

As a first step towards developing a dynamic model of the rat hindlimb, we measured muscle attachment and joint center coordinates relative to bony landmarks using stereophotogrammetry. Using these measurements, we analyzed muscle moment arms as functions of joint angle for most hindlimb muscles, and tested the hypothesis that postural change alone is sufficient to alter the function of selected muscles of the leg. We described muscle attachment sites as second-order curves. The length of the fit parabola and residual errors in the orthogonal directions give an estimate of muscle attachment sizes, which are consistent with observations made during dissection. We modeled each joint as a moving point dependent on joint angle; relative endpoint errors less than 7% indicate this method as accurate. Most muscles have moment arms with a large range across the physiological domain of joint angles, but their moment arms peak and vary little within the locomotion domain. The small variation in moment arms during locomotion potentially simplifies the neural control requirements during this phase. The moment arms of a number of muscles cross zero as angle varies within the quadrupedal locomotion domain, indicating they are intrinsically stabilizing. However, in the bipedal locomotion domain, the moment arms of these muscles do not cross zero and thus are no longer intrinsically stabilizing. We found that muscle function is largely determined by the change in moment arm with joint angle, particularly the transition from quadrupedal to bipedal posture, which may alter an intrinsically stabilizing arrangement or change the control burden.     

Analytical variables affecting exchangeable copper determination in blood plasma

To resolve discrepancies observed in the determination of plasma exchangeable Cu (also called direct reacting Cu or loosely bound Cu) by several methods, plasma storage techniques and various aspects of a stable isotope dilution procedure for exchangeable Cu were evaluated. Results indicated that the exchangeable Cu fraction of plasma increased with storage at room temperature, at 5°C and when subjected to repeated freeze/thaw cycles. Samples could be safely stored at −65°C. Exchange between added ⁶⁵Cu²⁺ and endogenous plasma Cu rapidly went to completion in the isotope dilution procedure. Analytical results were unaffected by shaking method, sample size or the presence of heparin. A small difference was observed between serum and plasma. The determination of exchangeable Cu did not vary over a period of 4 h when plasma was exposed to 1.6 × 10⁻⁴-M sodium diethyldithiocarbamate (used in the isotope dilution method) but steadily increased when exposed to 1.1 × 10⁻²-M sodium diethyldithiocarbamate, which suggested that tightly bound Cu (probably in ceruloplasmin) was exchanging with isotopic tracer at the higher concentration. Determination of exchangeable Cu was constant from pH 7.2–8.5 but increased substantially at higher pH. Complete recovery of natural Cu added to plasma was obtained. Studies in solution indicated that ⁶⁵Cu²⁺ exchanged readily with albumin- and amino acid-bound Cu. Ultrafiltration of plasma yielded a Cu fraction about half that of the exchangeable Cu fraction. We conclude that the stable isotope dilution procedure for plasma exchangeable Cu yields reliable, physiologically meaningful results.     

Analytical techniques for the determination of phenolic amine neurotransmitter conjugates

This paper reviews methods developed in the author's laboratory for the measurement of the two sulfate conjugates of dopamine in biological material. The methods are sensitive enough for most applications relating to dopamine sulfate. Some are applicable to measurement of sulfate conjugates of other phenolic amines.     

Comparative assessment of two algorithms for calibrating stereophotogrammetric systems

In this paper, a comparison is carried out between two photogrammetric algorithms aimed at camera calibration and three-dimensional target point reconstruction by ‘absolute’ distributions of control points; the first is Marzan and Karara's DLT, the second is CESNO, by the author, an algorithm quite close to Hatze's modified DLT (MDLT). The comparative assessment is especially aimed at testing the capability of the two methods to produce good results when calibration data are to be extrapolated beyond the space spanned by the control distribution. The assessment was carried out not by a real stereophotogrammetric system, but by the computer simulation of a two-camera set-up. Various combinations of internal camera parameters, such as the scaled principal distances, were tried out. As for the magnitude of the simulated non-linear lens distortion, three configurations were used which produced ‘low’, ‘medium’ and ‘strong’ distortion. The influence of decreasing the number of control (calibration) points on the accuracy performance of the two algorithms was also investigated. The results show the superiority of CESNO, especially with medium or strong lens distortion, or when the two camera principal distances sensibly differ.     

Limitations on cell volume determination using three-dimensional optical microscopy

One important application for three-dimensional microscopy is the determination of the volumes of biological cells. In this paper we investigate the application of three-dimensional deconvolution techniques to the problem of cell volume determination. The theoretical discussion is based on a Fourier sampling condition for reliable image reconstruction and it is shown, using this condition and simulated images, that the cell volume determination is reliable only for cells with a shape and orientation such that their length along the optic axis is much greater than their width.     

The three-dimensional determination of internal loads in the lower extremity

A three-dimensional model of the lower limb containing 47 muscles was developed to study the differences between a two- and three-dimensional approach for determining internal loads, the role of the dynamic joint representation, and the behavior of different load-bearing criteria in walking and running. The problem of redundancy of the musculo-skeletal system was resolved by applying inverse dynamics and static optimization methods. Different hypothetical load-bearing capabilities of hinge, spherical and intermediate joint types for the knee and the ankle joints were tested. It was found that even almost planar movements such as walking and running are associated with significant three-dimensional intersegment moments, especially in the frontal plane. Thus, a two-dimensional approach may underestimate internal loads up to 60%. It is shown that pure hinge joints are inappropriate for modeling the dynamical joint function of the knee and ankle joints. A more flexible joint representation in combination with a squared muscle stress minimization criterion predicted a lot of synergistic as well as antagonistic muscle activation which was also found in the EMG patterns. The results indicate the importance of muscular joint stabilization in natural human movements. Compared to in vivo measurements it is speculated that the predicted force magnitudes are considerably overestimated due to error propagation and still insufficient anatomical models. Thus, increased efforts to improve further the reliability of internal load calculations should be made in the future.