Radiofrequency radiation and the immune system. Part 3. In vitro effects on human immunoglobin and on murine T- and B-lymphocytes

Radiofrequency radiation (RFR) altered the physical separation of immunoglobulin (Ig) and of T- and B-lymphocytes during liquid gel chromatography. Exposure of human serum to a 10 MHz electric field (8500 V/m, less than or equal to 0.134 W kg-1) during chromatography resulted in accelerated elution of the IgM, IgA and IgG fractions. This effect is consistent with an increase in steric resistance of Ig molecules to the gel pores resulting in rapid elution. The low level of absorbed power employed did not lead to measurable heating of the gel media (25.00 +/- 0.05 degrees C). Effects on lymphocyte separation were investigated by performing immunoaffinity cell chromatography during exposure to 2500 MHz RFR (194 V/m, less than or equal to 0.117 W kg-1). Murine spleen lymphocytes were fractionated at 4.0 degrees C over Ig-derivatized agarose beads into Ig- and Ig+ lymphocyte subpopulations. RFR exposures resulted in premature elution of 19 per cent of the Ig+ (B-cell) population indicating an alteration of Ig binding. Temperature excursions in excess of +/- 0.05 degree C were not observed during exposures. These in vitro results demonstrate that Ig, whether freely diffusing in solution or bound to the lymphocyte cell surface, is influenced by RFR at absorbed power levels below the current recommended safety limit of 0.4 W kg-1 (U.S.A.). A possible mode of interaction for these effects as well as the relevance of these findings to in vivo biological processes are discussed.     

Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane

Dielectric breakdown of membranes of red blood cells was observed in high electric fields (approx. 10³–10⁴ V/cm) using an improved Coulter Counter with hydrodynamic focussing. In making measurements of the size distributions of red blood cells as a function of increasing electric field strenght it was found that a sharp discontinuity occurred in the otherwise linear relation between the pulse heights in the Coulter Counter and the electric field strength due to dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated at breakdown in the cell membranes yields a mean value of about 1.6 V for the membrane potential of red blood cells. Due to the dielectric break-down, release of hemoglobin occurred. Mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter or thermal rupture could be excluded as hemolysing mechanisms. The leaky ghost cells resealed at 37 °C as shown by incorporation of ¹³¹I-labeled albumin and repeated dielectric breakdown.     

Application of continuous zone electrophoresis to preparative separation of proteins

A comprehensive study of the application of continuous zone electrophoresis to preparative separation of proteins in free solution is presented. First, the influence of electric field strength, buffer residence time in the chamber, sample flow rate, and sample concentration on separation resolution and throughput were studied. Using multiple injections of sample into the electrophoresis chamber, a throughput of 500 mg protein/h was achieved for partially purified model proteins. Experiments on Escherichia coli crude extracts yielded a fivefold purification of beta-galactosidase along with a simultaneous separation of proteins from cell debris in a single step. Experiments correlating the electrophoretic mobility in continuous electrophoresis with the elution behavior in ion-exchange chromatography were performed on more than a dozen proteins which conclusively showed that separation of proteins in continuous zone electrophoresis is governed by net surface charge. Based on these results, the fraction numbers in which the proteins eluted could be correctly predicted. Proteins and enzymes with differences >0.5 M elution molarities in ion-exchange chromatography were separated by continuous zone electrophoresis on a preparative scale (mg/h or g/h) with >90% recovery. This corresponds to a preparative scale separation of proteins and enzymes which differ in apparent electrophoretic mobility by only 0.70 x 10(-5) cm(2)/V . s. (c) 1993 John Wiley & Sons, Inc.     

Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane.

Dielectric breakdown of membranes of red blood cells was observed in high electric fields (approx. 10-3-10-4 V/cm) using an improved Coulter Counter with hydrodynamic focussing. In making measurements of the size distributions of red blood cells as a function of increasing electric field strength it was found that a sharp discontinuity occurred in the otherwise linear relation between the pulse heights in the Coulter Counter and the electric field strength due to dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated at breakdown in the cell membranes yeilds a mean value of about 1.6 V. for the membrane potential of red blood cells. Due to the dielectric break-down, release of hemoglobin occurred. Mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter or thermal rupture could be excluded as hemolysing mechanisms. The leaky ghost cells resealed at 37 degrees C. as shown by incorporation of 131I-labeled albumin and repeated dielctric breakdown.     

Estimation of whole‐body SAR from electromagnetic fields using personal exposure meters

In this article, personal electromagnetic field measurements are converted into whole‐body specific absorption rates for exposure of the general public. Whole‐body SAR values calculated from personal exposure meter data are compared for different human spheroid phantoms: the highest SAR values (at 950 MHz) are obtained for the 1‐year‐old child (99th percentile of 17.9 µW/kg for electric field strength of 0.36 V/m), followed by the 5‐year‐old child, 10‐year‐old child, average woman, and average man. For the 1‐year‐old child, whole‐body SAR values due to 9 different radiofrequency sources (FM, DAB, TETRA, TV, GSM900 DL, GSM1800 DL, DECT, UMTS DL, WiFi) are determined for 15 different scenarios. An SAR matrix for 15 different exposure scenarios and 9 sources is provided with the personal field exposure matrix. Highest 95th percentiles of the whole‐body SAR are equal to 7.9 µW/kg (0.36 V/m, GSM900 DL), 5.8 µW/kg (0.26 V/m, DAB/TV), and 7.1 µW/kg (0.41 V/m, DECT) for the 1‐year‐old child, with a maximal total whole‐body SAR of 11.5 µW/kg (0.48 V/m) due to all 9 sources. All values are below the basic restriction of 0.08 W/kg for the general public. 95th percentiles of whole‐body SAR per V/m are equal to 60.1, 87.9, and 42.7 µW/kg for GSM900, DAB/TV, and DECT sources, respectively. Functions of the SAR versus measured electric fields are provided for the different phantoms and frequencies, enabling epidemiological and dosimetric studies to make an analysis in combination with both electric field and actual whole‐body SAR. Bioelectromagnetics 31:286–295, 2010. © 2009 Wiley‐Liss, Inc.     

Thermal and physiologic responses to 1200-MHz radiofrequency radiation: differences between exposure in E and H orientation

Ketamine-anesthetized Sprague-Dawley rats were exposed to far-field 1200-MHz continuous wave radiofrequency radiation in both E and H orientations (long axis of animal parallel to electric or magnetic field, respectively). Power densities were used that resulted in equivalent whole-body specific absorption rates of approximately 8 W/kg in both orientations (20 mW/cm2 for E and 45 mW/cm2 for H). Exposure was conducted to repeatedly increase colonic temperature from 38.5 to 39.5 degrees C in both orientations in the same animal. Irradiation in E orientation resulted in greater colonic, tympanic, left subcutaneous (side toward antenna), and tail heating. The results indicated a more uniform distribution of heat than that which occurred in previous experiments of 2450-MHz irradiation in E and H orientation. A lack of significant differences in blood pressure and heart rate responses between exposures in the two orientations in this study suggest that greater peripheral heating, as was seen in the earlier study of 2450 MHz, is necessary for these differences to occur.     

Frequency-dependent alterations in enolase activity in Escherichia coli caused by exposure to electric and magnetic fields

Some neurochemical effects of low-intensity electric and magnetic fields have been shown to be nonlinear functions of exposure parameters. These effects occurred within narrow ranges of frequency and intensity. Previous studies on membrane-associated endpoints in cell culture preparations demonstrated changes in calcium efflux and in acetylcholinesterase activity following exposure to radiofrequency radiation, amplitude modulated (AM) at 16 and at 60 Hz, at a specific absorption rate of 0.05 W/kg. In this study, these modulation frequencies were tested for their influence on the activity of a cytoplasmic enzyme, enolase, which is being tested clinically for detection of neoplasia. Escherichia coli cultures containing a plasmid with a mammalian gene for enolase were exposed for 30 min, and cell extracts were assayed for enolase activity by measuring absorbance at 240 nm. The enolase activity in exposed cultures was compared to the activity in paired control cultures. Exposure to 147 MHz carrier waves at 0.05 W/kg, AM at 16 Hz showed enolase activity enhanced by 62%, and AM at 60 Hz showed enolase activity reduced by 28%. Similarly, exposure to 16 Hz fields alone, at 21.2 V/m_rms (electric) and 97 nT_rms (magnetic), showed enhancement in enolase activity by 59%, whereas exposure to 60 Hz fields alone, at 14.1 V/m_rms (electric) and 65 nT_rms (magnetic), showed reduction in activity by 24%. Sham exposures as well as exposure to continuous-wave 147 MHz radiation at 0.05 W/kg showed no change in enolase activity. Although the underlying basis for these field effects in the cytoplasmic compartment has not been established, differential sensitivities to 16 Hz and to 60 Hz signals provide a clear focus for additional research to determine the responsible mechanism. © 1994 Wiley-Liss, Inc.     

Electrophoretic affinity chromatography: method validation

A new method for preparative-scale separation of biomolecules, electrophoretic affinity chromatography (EAC), is proposed in this paper. Separation by EAC is carried out in a long and ribbon-like multicompartment electrolyser separated by membranes, in which the two central compartments are used for packing the gel matrix and for sample loading respectively. Next to the central compartments are the elution compartments and electrode compartments. The electric field is applied perpendicular to the fluid flow in the compartments. Adsorption and desorption steps may both be carried out in the presence of an electric field, which transports the target components into the gel compartment for adsorption and the impurities into the elution compartments for washing. After the adsorption step an elution solution is introduced and the product is released from the gel matrix and washed out. Separation of human serum albumin (HSA) from human serum gives HSA product of high purity, as demonstrated by isoelectric focusing analysis. The characteristics of electrophoretic binding of HSA on Blue Sepharose Fast Flow are examined. The preliminary results show that this new method has advantages in terms of high rate of mass transfer and ease of scaling up, which are of particular interest when large-scale separation of biomolecules is considered.     

The antigenic structure ofFrancisella tularensis

The ether antigen ofFrancisella tularensis was fractionated using DEAE-cellulose and CM-cellulose chromatography, and Sephadex G-200 gel filtration. The CM-cellulose chromatography did not appear to be a suitable method for separation of individual components of the complex ether antigen. Most of the polysaccharide material and substances with a high phosphorus content were eluted already in the first peak of the elution curve. This method could be used only to separate a component, yielding one immunoelectrophoretic precipitin line, localized towards the cathode. On the other hand, the DEAE-cellulose chromatography, and particularly the Sephadex G-200 gel filtration (especially when recycling was introduced), yielded a clear separation of relatively clean components of the ether antigen. The present work provides a comparison between the immunochemical (immunoelectrophoretic) properties of fractions obtained by these methods and components isolated by salting out with ammonium sulphate, ethanol or trichloracetic acid precipitation.     

Characterization of macrophage activation factor, a lymphokine that causes macrophages to become cytotoxic for tumor cells.

Macrophage activation factor (MAF) was isolated from PPD-stimulated, BCG-immune mouse spleen cell culture fluids. In nine gel filtration runs on Sephadex G-100 or G-200, MAF was eluted in a single peak corresponding to a MW of 55,000 ± 1600. Recovery of activity was about 65%. Since the relative concentration curve of eluted MAF was wider than that of a single protein, MAF activity may be due to more than one protein with similar molecular weights. This possibility is strengthened by a broad elution range on DEAE cellulose chromatography, from a specific conductance of 3.5 to 8.5 mmho/cm, at pH 7.9. MAF was labile at both pH 4 and 10, and was destroyed by proteolytic enzymes. Eighty percent was destroyed by heating at 56 °C for 30 min. In affinity chromatography experiments, MAF did not bind to Con-A Sepharose; but it was bound to insolubilized Cibacron-blue and was eluted by an increase in ionic strength.     

Dynamic electric field assisted multi-dimensional liquid chromatography of biological samples

Complex biological samples require very high resolution separation strategies. The platform introduced here capitalises on the hyphenation of liquid chromatographic (LC) and electric potential gradient electrochromatographic multi-dimensional separation genres. First-dimension selectivity is provided by simultaneous size exclusion (SEC) and strong cation exchange (SCX) chromatography modes, while the second dimension comprises reversed phase (RP) characteristics in a dynamic (time-variant) electric field. The time-variant potential gradient with reversal of polarity is applied across the second dimension monolithic capillary throughout the duration of the solvent strength gradient elution. Hence, the platform offers comprehensive on-line sample clean-up (matrix depletion, analyte enrichment), fractionation (first dimention LC), and separation (second dimension LC) with the prospect of altering selectivity via polarity reversal dynamic electric field tuning.     

Dose-response relationship between body temperature and birth defects in radiofrequency-irradiated rats

Five groups of pregnant Sprague-Dawley rats were irradiated for 10-40 min on gestation day 9 in a 27.12-MHz radiofrequency field at a magnetic field strength of 55 A/m and an electric field strength of 300 V/m. The specific absorption rate was 10.8 +/- 0.3 W/kg. Exposures were terminated after the rat's colonic temperature reached 41.0 degrees C, 41.5 degrees C, 42.0 degrees C, 42.5 degrees C, or 43.0 degrees C. A control group was sham irradiated at 0 A/m and 0 V/m on gestation day 9, whereas a second control group was untreated. The incidence of both birth defects and prenatal death was directly related to maternal body temperature once a temperature threshold was exceeded. The temperature threshold for both types of effects was approximately 41.5 degrees C. A few pregnant rats died after exposure to 43.0 degrees C, and higher temperatures were nearly always lethal.     

Dielectric Breakdown of Cell Membranes

With human and bovine red blood cells and Escherichia coli B, dielectric breakdown of cell membranes could be demonstrated using a Coulter Counter (AEG-Telefunken, Ulm, West Germany) with a hydrodynamic focusing orifice. In making measurements of the size distributions of red blood cells and bacteria versus increasing electric field strength and plotting the pulse heights versus the electric field strength, a sharp bend in the otherwise linear curve is observed due to the dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated yields a value of about 1.6 V for the membrane potential at which dielectric breakdown occurs with modal volumes of red blood cells and bacteria. The same value is also calculated for red blood cells by applying the capacitor spring model of Crowley (1973. Biophys. J. 13:711). The corresponding electric field strength generated in the membrane at breakdown is of the order of 4 · 10⁶ V/cm and, therefore, comparable with the breakdown voltages for bilayers of most oils. The critical detector voltage for breakdown depends on the volume of the cells. The volume-dependence predicted by Laplace theory with the assumption that the potential generated across the membrane is independent of volume, could be verified experimentally. Due to dielectric breakdown the red blood cells lose hemoglobin completely. This phenomenon was used to study dielectric breakdown of red blood cells in a homogeneous electric field between two flat platinum electrodes. The electric field was applied by discharging a high voltage storage capacitor via a spark gap. The calculated value of the membrane potential generated to produce dielectric breakdown in the homogeneous field is of the same order as found by means of the Coulter Counter. This indicates that mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter could also be excluded as a hemolysing mechanism. The detector voltage (or the electric field strength in the orifice) depends on the membrane composition (or the intrinsic membrane potential) as revealed by measuring the critical voltage in E. coli B harvested from the logarithmic and stationary growth phases. The critical detector voltage increased by about 30% for a given volume on reaching the stationary growth phase.     

Method for the characterization of size-exclusion chromatography media for preparative purification of DNA restriction fragments

A protocol has been developed to characterize the potential of size exclusion chromatography media for the separation of DNA fragments at a preparative scale. A standard DNA mixture composed of -DNA cut with the restriction enzymes, AluI and HaeIII, was chromatographed on a column packed with the gel of interest. Fractions were collected and examined by PAGE. A digitized image of the gel was analyzed with the aid of a computer software program to determine the DNA fractionation range and selectivity curve for each chromatography gel. Four gels were characterized using this protocol. The effect of the elution buffer ionic strength on the fractionation of DNA was also investigated.The US Government right to retain a non-exclusive royalty-freelicense in and to any copyright is acknowledged.     

A Simple Biosynthetic Method for Preparation of High Specific Radioactivity Phosphatidyl-choline

Chemical changes in lysozyme during heating at 150~250°C for 20min were investigated by means of IR, ESR, and CD spectroscopies and gel permeation chromatography, and further a tryptic hydrolysate from the lysozyme heated at 200°C was analyzed by ion exchange chromatography. At 150°C, polymerization through disulfide linkages was observed, and at180°C, both polymerization and degradation occurred. When the temperature was raised to 200°C, remarkable changes in the structure of lysozyme, such as cleavage and recombination of peptide bonds, occurred. Over 200°C, polymerization and degradation occurred more violently.     

Erythrocyte hemolysis by radiofrequency fields

A field-strength-dependent hemolytic effect of continuous-wave radiofrequency (RF) exposure in vitro has been demonstrated. Erythrocytes in whole heparinized rabbit blood were hemolyzed by a 2-h exposure to 50- or 100-MHz RF fields at field strengths of greater than 4 V/cm. An effect of comparable magnitude resulted from exposure to 10-MHz RF at a field strength of 9 V/cm. Sample temperatures were maintained at 22.5 degrees +/- 0.2 degrees C. There was no apparent involvement of heating or temperature gradients, nor were there any RF exposure effects on cellular K+ or Na+ concentration, nor on pH. The mechanism of the hemolytic effect is not known. Since the percentage of lysed erythrocytes was less than 1% and there was an absence of effects on cellular cation concentrations, RF radiation may have irreversibly altered the plasma membrane permeability of a sensitive subpopulation of red cells (possibly aged cells) leading to osmotic lysis. RF radiation at these frequencies appears to affect red cells in a manner that is qualitatively and quantitatively different from microwave radiation.     

Multiplexed two-dimensional liquid chromatography for MALDI and nanoelectrospray ionization mass spectrometry in proteomics

We developed a multiplexed two-dimensional separation system based on reversed phase (RP)--strong cation exchange (SCX) chromatography as a front-end device for matrix-assisted laser desorption ionization (MALDI) or nanoelectrospray ionization (nanoESI) mass spectrometry. Tryptic peptide mixtures were fractionated on a reversed-phase HPLC column, and each fraction was loaded onto multiplexed SCX microcolumns. Because this second chromatography was carried out in parallel, the analysis time is independent of the fraction number in the first RP-HPLC separation. The resultant samples were desalted/concentrated and eluted onto a MALDI plate with matrix-containing elution solutions in parallel, or eluted with optimized solutions for nanoESI and loaded onto nanoESI sprayers by an automated instrument. The soluble portion of HCT116 lysate was digested and fractionated using a 48-plexed chromatography system. Approximately 1000 unique peaks were detected in MALDI-MS with 3000 MS/MS spectra, while 724 peptides with ultrahigh peptide mass accuracy (sub-ppm error) were identified in nanoESI-FTICR mass spectrometry with five integrated selected ion monitoring scans. Since MS measurement with this off-line LC-LC approach is not restricted by continuous LC elution, it is expected to be useful especially in cases where repeated analysis with different scan modes or long-term data acquisition is required.     

Multiple power-density windows and their possible origin

We have previously reported that in vitro exposure of chick forebrain tissue to 50-MHz radiofrequency (RF) electromagnetic radiation, amplitude modulated (AM) at 16 Hz, would enhance the efflux of calcium ions within only two power-density ranges: one from 1.44 to 1.67 mW/cm2, and the other including 3.64 mW/cm2. No effect on efflux occurred at 0.37, 0.72, 2.17, and 4.32 mW/cm2. We confirmed and extended these results by testing at another set of power densities, which included the range of the previous study. Forebrain tissue from 1-7-day-old chickens was labeled in vitro with radioactive calcium ions (30 min, at 37 degrees C), rinsed, placed in a physiological salt solution, and then exposed for 20 min to 50-MHz radiation, AM at 16 Hz, in a transverse electric and magnetic field (TEM) cell maintained at 37 degrees C. The solution was then assayed for radioactive calcium activity. A power-density series was tested. An enhanced efflux of calcium ions was found at 1.75, 3.85, 5.57, 6.82, 7.65, 7.77, and 8.82 mW/cm2; no change was observed at 0.75, 2.30, 4.50, 5.85, 7.08, 8.19, 8.66, 10.6, and 14.7 mW/cm2. Power density is converted to specific absorption rate (SAR) by 0.36 mW/kg per mW/cm2. Even the highest SAR tested (0.005 W/kg) is much too low to result in generalized heating of the sample and thus to be the underlying cause of the enhanced response. A hypothetical mechanism is proposed involving dynamic systems that may account for the power-density dependency as well as for part of the frequency dependency observed with both modulated RF radiation and extremely-low-frequency (ELF) fields.     

螺旋槽圆盘柱高速逆流色谱及其在多肽和蛋白分离中的应用

本研究重点考察了实验室自行设计研制的J型螺旋槽圆盘柱逆流色谱系统对正丁醇-醋酸-水体系和聚乙二醇(PEG1000)-磷酸盐-水双水相体系的固定相保留能力,并研究了流动相流速(F)、柱转速(w)和温度(T)等因素对固定相保留率(Sf)的影响。结果表明,该新型分离柱可使两种溶剂体系在L-I-T、U-O-H和L-I-H三种洗脱模式下都可获得较高的Sf,即以下相为流动相,采用由螺旋槽内端(I)向外端(O)的流通方式,或以上相为流动相,采用由螺旋槽外端(O)向内端(I)的流通方式可以获得较高的固定相保留。其保留能力较传统的螺旋管逆流色谱柱有显著提高。Sf随着w的增加而升高,随着F的增加而降低,且Sf与F1/2/w线性相关。20oC～45oC之间温度对Sf影响不明显,但低于20oC不利于双水相体系的保留。应用研究表明,采用正丁醇-醋酸-水(4:1:5,V/V/V)和PEG1000-磷酸钾盐-水(12.5:12.5:75,W/W/W)(pH9.0)体系可以在较高的流动相流速和较高的固定相保留下分别实现对亮氨酸-酪氨酸(Leu-Tyr)和缬氨酸-酪氨酸(Val-Tyr)二肽混合物、细胞色素C与肌红蛋白混合物、肌红蛋白与溶菌酶...     

Summary of measured radiofrequency electric and magnetic fields (10 kHz to 30 GHz) in the general and work environment

We have plotted data from a number of studies on the range of radiofrequency (RF) field levels associated with a variety of environmental and occupational sources. Field intensity is shown in units of volts/meter (V/m) for electric field strength and amps/meter (A/m) for magnetic field strength. Duty factors, modulation frequencies, and modulation indices are also reported for some sources. This paper is organized into seven sections, each cataloging sources into appropriate RF frequency bands from very-low frequency (VLF) to super-high frequency (SHF), and covers frequencies from 10 kHz to 30 GHz. Sources included in this summary are the following: Coast Guard navigational transmitters, a Navy VLF transmitter, computer visual display terminals (VDTs), induction stoves or range tops, industrial induction and dielectric heaters, radio and television broadcast transmitters, amateur and citizens band (CB) transmitters, medical diathermy and electrosurgical units, mobile and handheld transmitters, cordless and cellular telephones, microwave ovens, microwave terrestrial relay and satellite uplinks, and police, air traffic, and aircraft onboard radars. For the sources included in this summary, the strongest fields are found near industrial induction and dielectric heaters, and close to the radiating elements or transmitter leads of high power antenna systems. Handheld transmitters can produce near fields of about 500 V/m at the antenna. Fields in the general urban environment are principally associated with radio and TV broadcast services and measure about 0.1 V/m root-mean-square (rms). Peak fields from air traffic radars sampled in one urban environment were about 10 V/m, 300 times greater than the rms value of 0.03 V/m when the duty factor associated with antenna rotation and pulsing are factored in. Bioelectromagnetics 18: 563–577, 1997. Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.