Non-Thermal Radio Frequency and Static Magnetic Fields Increase Rate of Hemoglobin Deoxygenation in a Cell-Free Preparation

The growing body of clinical and experimental data regarding electromagnetic field (EMF) bioeffects and their therapeutic applications has contributed to a better understanding of the underlying mechanisms of action. This study reports that two EMF modalities currently in clinical use, a pulse-modulated radiofrequency (PRF) signal, and a static magnetic field (SMF), applied independently, increased the rate of deoxygenation of human hemoglobin (Hb) in a cell-free assay. Deoxygenation of Hb was initiated using the reducing agent dithiothreitol (DTT) in an assay that allowed the time for deoxygenation to be controlled (from several min to several hours) by adjusting the relative concentrations of DTT and Hb. The time course of Hb deoxygenation was observed using visible light spectroscopy. Exposure for 10–30 min to either PRF or SMF increased the rate of deoxygenation occurring several min to several hours after the end of EMF exposure. The sensitivity and biochemical simplicity of the assay developed here suggest a new research tool that may help to further the understanding of basic biophysical EMF transduction mechanisms. If the results of this study were to be shown to occur at the cellular and tissue level, EMF-enhanced oxygen availability would be one of the mechanisms by which clinically relevant EMF-mediated enhancement of growth and repair processes could occur

Effect of a pulsed radiofrequency electromagnetic field on in vitro cell-free nitric oxide synthesis

An in vitro, cell free nitric oxide assay was developed to assess the effect of a pulsed radiofrequency electromagnetic field (EMF, 27.12 MHz) on enzymatic synthesis. Calcium calmodulin-dependent synthesis via neuronal and endothelial nitric oxide synthases was measured using the hemoglobin assay. No changes in NO synthesis were observed for EMF vs. ambientonly exposure

Non-thermal electromagnetic fields increase rate of hemoglobin deoxygenation in a cell-free preparation

The reducing agent dithiothreitol was used to deoxygenate human haemoglobin, and the time course observed via visible light spectroscopy. A decrease in the time required for deoxygenation was found for exposures to both a pulsed radiofrequency signal currently in clinical use for treatment of pain and edema, and a 150 mT static magnetic field

EMF effect on hemoglobin (Hb) visible light spectra.

<p>Spectra representative of the effects of pulsed radiofrequency (PRF) signal and 186 mT static magnetic field (SMF) on Hb visible light spectra during bHb)deoxygenation. Data shown are typical of samples drawn from a single tube for each EMF exposure condition, shown here at 150 min after a single 30 min EMF exposure. Deoxygenation of 100 µM Hb was carried out in 50 mM Hepes buffer (pH 7.2) using the reducing agent dithiothreitol (20 mM) at 22°C, and is characterized here by the passage of the spectrum from a two-peaked to one-peaked form. Deoxygenation occured at an earlier time for EMF exposed samples (traces with one peak), as compared to control samples exposed only to the ambient geomagnetic laboratory environment (trace with two peaks). The EMF effect was observable at the time of most rapid deoxygenation.</p

Effect of the static magnetic field on time course of hemoglobin deoxygenation.

<p>Hb deoxygenation after 10 min exposure to 186 mT static magnetic field (SMF) in 5M urea. No significant change in oxy/deoxy ratio was visible until the time of rapid deoxygenation, at approximately 40 min. The time of most rapid deoxygenation occurred approximately 10 min earlier for SMF treated samples (P<0.002 for 42 min</p

Addition of 5 M urea to reaction mixture.

<p>Addition of 5 M urea to the deoxygenation assay reduces the time required for deoxygenation and render the PRF effect more apparent. The maximal difference between PRF-treated and control samples occurred at 73 min, with PRF-treated samples showing a significant (70.4±9.7)% reduction in oxyHb concentration, as compared to controls (8.5±2.1 µM vs. 28.8±6.3 µM, P<0.03, n = 5), in contrast to the 30.8% reduction observed in the absence of urea (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061752#pone-0061752-g002" target="_blank">Figure 2</a>).</p

Reaction plate showing that addition of 5M urea to reaction solution renders magnetic field effect more apparent.

<p>96-well spectrophotometer plate shown at 46 min after initiation of reaction of hemoglobin with dithiothreitol (DTT). Samples treated for 10 min with 186 mT static magnetic field (3 left-hand columns in plate) substantially completed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061752#pone-0061752-g004" target="_blank">Figure 4</a>) DTT-induced deoxygenation before untreated samples began to lose O₂, rendering the magnetic field effect visibly apparent.</p

Effect of the pulsed radiofrequency field on time course of hemoglobin deoxygenation.

<p>Time course of oxy hemoglobin (HbO₂) concentration, after a single 30 min pulsed radiofrequency electromagnetic field (PRF) exposure of Hb, under deoxygenating conditions. Concentration was determined by visible light spectroscopy at 560, 576, 630 nm. PRF exposure resulted in a significant (30.8±10.3)% reduction in oxyHb concentration, as compared to controls (14.6±1.3 µM vs. 21.1±2.5 µM, P<0.03, n = 5), suggesting an alteration in Hb solution properties that persisted after PRF signal was removed.</p