Osmolality dependence of erythrocyte sedimentation and aggregation in a strong magnetic field

In order to specify the major determinant of the magnetic enhancement of erythrocyte sedimentation observed previously, the dependence of erythrocyte sedimentation rate (ESR) on osmolality was measured under a strong magnetic field. Even at hypotonic osmolality, an increase in ESR due to aggregation was observed in plasma solution as compared with that without aggregation in saline solution. However, the magnetic field did not enhance ESR at hypotonic osmolality, when the cell shape was an isotropic sphere (spherocyte). Thus, we narrowed our search to a mechanism that would explain the enhanced ESR found specifically in anisotropic erythrocytes. It was concluded that the major determinant can only work for anisotropic erythrocytes and is a magnetic field-induced increase in an intermembrane adhesive area due to magnetic orientation of anisotropic erythrocytes.     

Effects of an external magnetic field on the flock size and sedimentation of activated sludge

Effects of an external magnetic field on sedimentation enhancement were investigated using three kinds of practical activated sludge. An indoor experiment was setup (3.5l aeration vessel) equipped with a broth circulation system and 800 G of external magnetic field. The application of an external magnetic field to the activated sludge enhanced the sedimentation irrespective of the nature of the activated sludge. At the same time, the flock size of the sludge was enlarged by the external magnetic field. However, the surface zeta potentials of the sludge were not changed by the external magnetic field. Addition of FeCl₃ to the sludge enhanced the effects of the external magnetic field. Based on these results, the following mechanism for the enhancement of sedimentation by the external magnetic field is suggested: activated sludge containing iron was magnetized during entry to the magnetic field and coagulated with the magnetic force. As a consequence, the flock size was enlarged. Almost the same sedimentation enhancement by external magnetization was confirmed by a pilot scale magnetization system (2.0 m³ of aeration vessel) when the airlift type of mild broth circulation system was equipped with a 5000 G magnetic field.     

Rapid, high-yield purification of cell surface membrane using colloidal magnetite coated with polyvinylamine: sedimentation versus magnetic isolation

A new technique for the magnetic isolation of external plasma membrane from Dictyostelium discoideum is described and compared to a previously published procedure employing sedimentation of silica-coated plasma membrane. The magnetic isolation technique involves coating intact cells with a polyvinylamine-magnetite colloid and overcoating with polyacrylate to form a dense pellicle. The magnetite pellicle totally coated the cells and was not internalized. Coated cells were lysed and membrane fragments retrieved from the cell homogenate using a diverging field electromagnet. The membrane obtained in such a manner was analyzed for marker enzyme activity and cell surface label. The plasma membrane was obtained in high yield (42%) with an average purification of 8-fold. The polyvinylamine-magnetite pellicle shielded the external plasma membrane face to proteolysis by papain and pronase. It also acted as a barrier to alpha-methylmannoside in concanavalin A-carbohydrate competition studies.     

In vivo interaction between nitrogenase molybdenum-iron protein and membrane in Azotobacter vinelandii and Rhodospirillum rubrum

Oriented whole cell multilayers of Azotobacter vinelandii and Rhodospirillum rubrum were analyzed by electron spin resonance (ESR) spectroscopy to detect possible structural associations between nitrogenase molybdenum-iron (MoFe) protein and cytoplasmic or intracytoplasmic membrane. Initially, protocols were designed to obtain strong molybdenum-iron protein ESR signals in whole cell samples of each organism. Then, two-dimensional orientation of whole cell membranes was demonstrated in whole cell multilayers using doxyl stearate spin label in A. vinelandii and the bacteriochlorophyll a dimer triplet signal, (BCHl a)T2, from the intracytoplasmic membrane-bound photosynthetic apparatus of R. rubrum. Subsequent analysis of the low-field signals, g = 4.3 and g = 3.6, of molybdenum-iron protein in whole cell multilayers of each organism showed orientation-dependent characteristics, although the properties of each were different. Specifically, as the normal to the membrane plane was rotated from perpendicular to parallel with the ESR magnetic field, the amplitude of the g = 3.6 signal decreased from maximum to about 37% of maximum in A. vinelandii and from maximum to about 88% of maximum in R. rubrum. The angular dependence of the g = 4.3 peak during rotation varied in A. vinelandii, but decreased from maximum to about 63% of maximum in R. rubrum. These data suggest that the molybdenum-iron protein of nitrogenase was oriented in response to the physical orientation of cellular membranes and that a structural association may exist between this nitrogenase component and membrane in these organisms.     

A physical theory of erythrocyte sedimentation

A new physical theory of erythrocyte sedimentation is proposed. Various assumptions underlying Stokes' formula are first criticized. An explicit formula is proposed, taking into account some of the results of recent experimental investigations including the effect of upward flow of plasma and the time course of growth of aggregates. It is generally shown that the sedimentation curve without aggregation never becomes a sigmoid. Our formula is applicable to the increased ESR due to the aggregation of erythrocytes. The sedimentation velocity depends not only on the hematocrit and the ultimate size of the aggregates, but also on the retardation time of the growth of aggregates in conformity with the experimental result of Kernick et al.     

Structure and flexibility of plasma fibronectin in solution: electron spin resonance spin-label, circular dichroism, and sedimentation studies

Human plasma fibronectin has been investigated by electron spin resonance (ESR) spin-label methods in conjunction with circular dichroism (CD) and sedimentation techniques to investigate its structure and flexibility in solution. The buried sulfhydryl groups of fibronectin were modified with a maleimide spin-label [Lai, C.-S., & Tooney, N. M. (1984) Arch. Biochem. Biophys. 228, 465-473]. Both conventional and saturation transfer ESR spectra give a rotational correlation time of about (2-3) X 10(-8) s for plasma fibronectin, a value that is at least 40 times faster than the rotational correlation time calculated from the minimal molecular dimensions. This argues that plasma fibronectin is not a compact, globular protein and suggests that the regions of ordered structural domains have a relatively high degree of independent mobility. ESR, CD, and sedimentation measurements showed that many structural features of plasma fibronectin remain unchanged when the pH is decreased from 7.4 to 3.0. On the other hand, ESR results indicate an unfolding of the protein molecule either at pH 11 or in 4 M urea solution. Similarly, the sedimentation coefficient decreases from about 13 to 8.4 S when the pH is raised to 10.8. At pH values above 11, the CD spectrum resembles a random coil; however, some ordered structure is retained either at pH 11 or in 4 M urea. It is likely that the sulfhydryl-containing regions of the molecule are more sensitive to urea or alkali than are portions of the molecule stabilized by intrachain disulfide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Graded alterations of RBC aggregation influence in vivo blood flow resistance

Although the effects of red blood cell (RBC) aggregation on low-shear rate blood viscosity are well known, the effects on in vivo flow resistance are still not fully resolved. The present study was designed to explore the in vivo effects of RBC aggregation on flow resistance using a novel technique to enhance aggregation: cells are covalently coated with a block copolymer (Pluronic F-98) and then suspended in unaltered plasma. RBC aggregation was increased in graded steps by varying the Pluronic concentration during cell coating and was verified by microscopy and erythrocyte sedimentation rate (ESR), which increased by 200% at the highest Pluronic level. RBC suspensions were perfused through an isolated in situ guinea pig hindlimb preparation while the arterial perfusion pressure was held constant at 100 mmHg via a pressure servo-controlled pump. No significant effects of enhanced RBC aggregation were observed when studies were conducted in preparations with intact vascular control mechanisms. However, after inhibition of smooth muscle tone (using 10(-4) M papaverin), a significant change in flow resistance was observed in a RBC suspension with a 97% increase of ESR. Additional enhancements of RBC aggregation (i.e., 136 and 162% increases of ESR) decreased flow resistance almost to control values. This was followed by another significant increase in flow resistance during perfusion with RBC suspensions with a 200% increase of ESR. This triphasic effect of graded increases of RBC aggregation is most likely explained by an interplay of several hemodynamic mechanisms that are triggered by enhanced RBC aggregation.     

Blood viscosity parameter correlation with types of leukemia.

We investigated the hemorheological, hematological and biochemical parameters in 30 cases of acute lymphocytic leukemia (ALL), 21 cases of acute myelogenous leukemia (AML) and 30 cases of chronic myelogenous leukemia (CML). The parameters studied include whole blood viscosity, plasma viscosity, erythrocyte sedimentation rate (ESR), red cell filterability, hematocrit, platelet count and aggregation, fibrinogen, hemoglobin, leucocyte count, bleeding time and lactate dehydrogenase activity (LDH). In the cases of ALL we observed significant decrease in whole blood viscosity, hemoglobin, hematocrit and platelet count but an increase in plasma viscosity, fibrinogen, bleeding time and LDH activity. In the cases of AML, we observed increase in whole blood viscosity, plasma viscosity, ESR, fibrinogen, leucocyte count, bleeding time and LDH activity but decrease in the hemoglobin, hematocrit and platelet count. In the cases of CML, we observed an increase of whole blood viscosity, plasma viscosity, ESR, fibrinogen elevation but decreases in bleeding time. In all cases, red cell filterability was unaffected.     

血浆是引起血沉增快的主要因素

目的：阐明血沉增快的原因是血浆还是红细胞。方法：收集72例血沉异常的抗凝血标本,同时收集血型相对应的72例血沉正常标本,组成血型相同的血沉异常和正常标本72对,每对互换血浆后重新测定血沉,与原始血沉结果比较,并通过多元线性回归分析血沉与血浆蛋白及血脂浓度的关系。结果：血沉异常标本的红细胞加入血沉正常标本的血浆后,72例（100%）血沉均减慢,其中30例血沉下降90%以上,35例下降70%-90%,7例小于70%。血沉正常标本的红细胞加入血沉异常标本的血浆后,67例（93%）血沉加快,其中58例（81%）变为异常（18例血沉加快10倍以上,40例加快5-10倍）。球蛋白、白蛋白和纤维蛋白原与血沉具有线性关系,球蛋白（r=0.420,P〈0.001）和纤维蛋白原（r=0.673,P〈0.001）与血沉呈正相关,而白蛋白（r=-0.558,P〈0.001）与血沉呈负相关。结论：血沉增快主要与血浆因素相关,红细胞对于血沉的影响作用很小。     

Action of a 50 Hz magnetic field on proliferation of cells in culture

Proliferation of SV40-3T3 mouse fibroblasts and human HL-60 promyelocytes was studied after treatment with a sinusoidal 2 mT_rms 50 Hz magnetic field. A single exposure of 60 minutes caused quasicyclic changes in the cell number of SV40-3T3 cultures as function of time after treatment, which was interpreted to be due to the induction of chronobiological mechanisms by the field. Moreover, small variations in cell cycle distribution were measured during postexposure incubation for both cell lines. To discriminate between the effect of the magnetic vector and the induced electric field, HL-60 cell exposure was also performed on organ culture dishes. These dishes consist of two coaxially centered, isolated compartments in which different electric field levels are induced in the medium during treatment. Cell growth was affected in the outer compartment only where the induced electric field ranged from 8 to 12 mV_peak/meter at 2 mT, but it was not affected in the inner compartment (field range 0–4 mV_peak/meter). This suggests that the effects on cell growth are due to the induced electric field and are expressed only above a threshold of between 4 and 8 mV_peak/meter. Bioelectromagnetics 18:177–183, 1997. © 1997 Wiley-Liss, Inc.     

ESR imaging of myelin basic protein induced vesicle aggregation

Using a modulated magnetic field gradient technique, the conventional ESR spectrum of well-defined spatial sections and the one-dimensional-ESR image of the nitroxide centre line of spin-labeled stearic acid in phospholipid vesicles were recorded with a spatial resolution of 4.10(-5) m after pelleting the vesicles inside 1 mm (i.d.) sample capillaries in a slow centrifuge (2500 X g). The sedimentation characteristics of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol vesicles were quantitatively compared with particular reference to vesicle aggregation induced by myelin basic protein. Protein-induced changes in the effective molecular mass were determined from ESR images of sedimentation profiles. The present data lend further support to the notion that the primary target of myelin basic protein-lipid interaction is the acidic lipid pool of myelin.     

Effects of inflammatory disease on plasma oxprenolol concentrations.

When single oral doses of oxprenolol were given to three healthy subjects on three separate occasions under standardised conditions the plasma concentration-time curves for each subject were closely similar. In two of the subjects, however, a mild illness led to a dramatic, temporary increase in the peak plasma concentration and area under the plasma concentration-time curve (AUC). This effect of inflammatory disease was confirmed by comparing a group of patients with an erythrocyte sedimentation rate (ESR) of over 20 mm in the first hour with a group whose ESR was below this value. The mean peak plasma concentration and AUC were significantly higher in the group with a raised ESR. This may be related to altered concentrations of one of the acute-phase proteins. Thus it is concluded that plasma oxprenolol concentrations are raised in inflammatory disease, but further work is needed to determine the mechanism of this increase.     

Effects of Aggregation on Blood Sedimentation and Conductivity

The erythrocyte sedimentation rate (ESR) test has been used for over a century. The Westergren method is routinely used in a variety of clinics. However, the mechanism of erythrocyte sedimentation remains unclear, and the 60 min required for the test seems excessive. We investigated the effects of cell aggregation during blood sedimentation and electrical conductivity at different hematocrits. A sample of blood was drop cast into a small chamber with two planar electrodes placed on the bottom. The measured blood conductivity increased slightly during the first minute and decreased thereafter. We explored various methods of enhancing or retarding the erythrocyte aggregation. Using experimental measurements and theoretical calculations, we show that the initial increase in blood conductivity was indeed caused by aggregation, while the subsequent decrease in conductivity resulted from the deposition of erythrocytes. We present a method for calculating blood conductivity based on effective medium theory. Erythrocytes are modeled as conducting spheroids surrounded by a thin insulating membrane. A digital camera was used to investigate the erythrocyte sedimentation behavior and the distribution of the cell volume fraction in a capillary tube. Experimental observations and theoretical estimations of the settling velocity are provided. We experimentally demonstrate that the disaggregated cells settle much slower than the aggregated cells. We show that our method of measuring the electrical conductivity credibly reflected the ESR. The method was very sensitive to the initial stage of aggregation and sedimentation, while the sedimentation curve for the Westergren ESR test has a very mild slope in the initial time. We tested our method for rapid estimation of the Westergren ESR. We show a correlation between our method of measuring changes in blood conductivity and standard Westergren ESR method. In the future, our method could be examined as a potential means of accelerating ESR tests in clinical practice.     

Dosimetry in magnetic fields with dedicated MR-compatible ionization chambers

MR-integrated radiotherapy requires suitable dosimetry detectors to be used in magnetic fields. This study investigates the feasibility of using dedicated MR-compatible ionization chambers at MR-integrated radiotherapy devices. MR-compatible ionization chambers (Exradin A19MR, A1SLMR, A26MR, A28MR) were precisely modeled and their relative response in a 6MV treatment beam in the presence of a magnetic field was simulated using EGSnrc. Monte Carlo simulations were carried out with the magnetic field in three orientations: the magnetic field aligned perpendicular to the chamber and beam axis (transverse orientation), the magnetic field parallel to the chamber as well as parallel to the beam axis. Monte Carlo simulation results were validated with measurements using an electromagnet with magnetic field strength upto 1.1 T with the chambers in transverse orientation. The measurements and simulation results were in good agreement, except for the A26MR ionization chamber in transverse orientation. The maximum increase in response of the ionization chambers observed was 8.6% for the transverse orientation. No appreciable change in chamber response due to the magnetic field was observed for the magnetic field parallel to the ionization chamber and parallel to the photon beam.Polarity and recombination correction factor were experimentally investigated in the transverse orientation. The polarity effect and recombination effect were not altered by a magnetic field.This study further investigates the response of the ionization chambers as a function of the chambers’ rotation around their longitudinal axis. A variation in response was observed when the chamber was not rotationally symmetric, which was independent of the magnetic field.     

Effects of low frequency electric fields on the sedimentation rate of human blood. Preliminary observations

The erythrocyte sedimentation rate (ESR) of human blood samples exposed to electric fields at frequencies ranging from 1 Hz to 100 kHz was measured. Statistically significant differences in ESR were found between the exposed and the control samples at most of the frequencies used. The results suggest a possible nonlinear dependence of the effect on frequency, the threshold field strength varying from about 25 V/m to about 5 kV/m.     

Fluorescence imaging in the millisecond time range of membrane electropermeabilisation of single cells using a rapid ultra-low-light intensifying detection system

A fast and sensitive fluorescence image acquisition system is described which uses an ultra-low-light intensifying camera able to acquire digitised fluorescence images with a time resolution of 3.33 ms/image. Two modes of recording were employed. The synchronisation mode allowed acquisition of six successive 3.33 ms-images synchronised with an external trigger, while the memorisation mode allowed acquisition of twelve successive 3.33 ms images starting after a 20 ms-time lag from the external trigger. Interaction of ethidium bromide (EB) with the membrane of electropermeabilised living cells was studied using this imaging system. We observed enhanced fluorescence of the dye when associated with electropermeabilised cells. Using single cells, 3.33 ms-images of the fluorescence interaction patterns of ethidium bromide showed well-defined membrane labelling. The enhanced fluorescence patterns were shown to represent the electropermeabilised area of the cell membrane. The average level of fluorescence associated with the labelled part of the cell membrane increased linearly during and immediately (less than 7 ms) after the electropermeabilisation pulse. Steady-state EB interaction with the membrane was achieved in a maximum 20 ms-time lag after electropermeabilisation. The membrane labelled parts were always observed in the cell regions facing the electrodes. They were present only when the electric field strength was higher than a threshold value which was different for the two cell sides. An increase in electric field intensity led to an increase in the dimensions of the labelled cell region. Received: 7 August 1997 / Revised version: 14 November 1997 / Accepted: 15 January 1998     

Orientation of paramecium swimming in a DC magnetic field

We found that a ciliated protozoan, Paramecium, swam perpendicular to a static (DC) magnetic field (0.68 T). The swimming orientation was similar even when the ionic current through the cell membrane disappeared after saponin treatment. To determine the diamagnetic anisotropy of intracellular organs, macronuclei, cilia, and secretory vesicles, trichocysts, were selectively isolated. Both cilia and trichocysts tended to align their long axis parallel to the magnetic field (0.78 T). Paramecium mutants that lack trichocysts also swam perpendicular to the magnetic field, although the proportion fraction was smaller than the normal population. Since large numbers of cilia and trichocysts are arranged at right angles to the long axis of the cell, the diamagnetic anisotropies of cilia and trichocysts cause the long axis of the cell to align perpendicular to the magnetic field. In contrast to the DC magnetic field, an alternative (AC) magnetic field (60 Hz, 0.65 T) had almost no effect on the swimming orientation of Paramecium.     

Reconstruction of the probe angular distribution from a series of electron spin resonance spectra of tilted oriented samples.

Model-independent methods for the reconstruction of the nitroxide spin probe angular distribution of labeled oriented biological assemblies from electron spin resonance (ESR) spectra were investigated. We found that accurate probe angular distribution information could be obtained from the simultaneous consideration of a series of ESR spectra originating from a sample at differing tilt angles relative to the Zeeman magnetic field. Using simulated tilt series data sets, we developed a consistent criteria for judging the reliability of the simulated fit to the data as a function of the free spectral parameters and thereby have increased the significance of the model-independent reconstruction of the probe angular distribution derived from the fit. We have also enhanced the angular resolution measurable with the model-independent methodology by increasing the rank of the order parameters that we can reliably deduce from a spectrum. This enhancement allows us to accurately deduce higher resolution features of the spin probe distribution. Finally we investigated the usefulness of fitting the tilt series data in multiple data sets such that tilt series data from many identical sample preparations are fitted simultaneously. This method proved to be useful in rapidly reducing a large amount of data by eliminating any redundant computations in the application of the enhanced model-independent analysis to identical sets of tilt series data. We applied the methodology developed here to ESR spectra from probe labeled muscle fibers to study the orientation of myosin cross-bridges in fibers. This application is described in the accompanying paper.     

The role of temporal sensing in bioelectromagnetic effects

Experiments were conducted to see whether the cellular response to electromagnetic (EM) fields occurs through a detection process involving temporal sensing. L929 cells were exposed to 60 Hz magnetic fields and the enhancement of ornithine decarboxylase (ODC) activity was measured to determine cellular response to the field. In one set of experiments, the field was turned alternately off and on at intervals of 0.1 to 50 s. For these experiments, field coherence was maintained by eliminating the insertion of random time intervals upon switching. Intervals ≥ 1 s produced no enhancement of ODC activity, but fields switched at intervals ≥ 10 s showed ODC activities that were enhanced by a factor of approximately 1.7. These data indicate that it is the interval over which field parameters (e.g., amplitude or frequency) remain constant, rather than the interval over which the field is coherent, that is critical to cellular response to an EMF. In a second set of experiments, designed to determine how long it would take for cells to detect a change in field parameters, the field was interrupted for brief intervals (25–200 ms) once each second throughout exposure. In this situation, the extent of EMF-induced ODC activity depended upon the duration of the interruption. Interruptions ≥ 100 ms were detected by the cell as shown by elimination of field-induced enhancement of ODC. That two time constants (0.1 and 10 s) are involved in cellular EMF detection is consistent with the temporal sensing process associated with bacterial chemotaxis. By analogy with bacterial temporal sensing, cells would continuously sample and average an EM field over intervals of about 0.1 s (the “averaging” time), storing the averaged value in memory. The cell would compare the stored value with the current average, and respond to the EM field only when field parameters remain constant over intervals of approximately 10 s (the “memory” time). Bioelectromagnetics 18:388–395, 1997. © 1997 Wiley-Liss, Inc.     

Applied DC magnetic fields cause alterations in the time of cell divisions and developmental abnormalities in early sea urchin embryos

Most work on magnetic field effects focuses on AC fields. The present study demonstrates that exposure to medium-strength (10 mT-0.1 T) static magnetic fields can alter the early embryonic development of two species of sea urchin embryos. Batches of fertilized eggs from two species of urchin were exposed to fields produced by permanent magnets. Samples of the continuous cultures were scored for the timing of the first two cell divisions, time of hatching, and incidence of exogastrulation. It was found that static fields delay the onset of mitosis in both species by an amount dependent on the exposure timing relative to fertilization. The exposure time that caused the maximum effect differed between the two species. Thirty millitesla fields, but not 15 mT fields, caused an eightfold increase in the incidence of exogastrulation in Lytechinus pictus, whereas neither of these fields produced exogastrulation in Strongylocentrotus purpuratus. Bioelectromagnetics 18:255–263, 1997. © 1997 Wiley-Liss, Inc.