Nonionizing radiation effect on the markup of blood cells with 99 mtc in vitro

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NON-IONIZING RADIATION EFFECT ON THE MARKUP OF BLOOD CELLS WITH 99

MTC IN VITRO

Carlos Ricardo de Queiroz Martiniano¹, Lídia Audrey Rocha Valadas Marques¹, Edilson Martins Rodrigues

Neto

, Mara Assef Leitão Lotif¹, Érika Sabóia Guerra Diógenes¹, Allana Bezerra Capistrano¹, Mayra Furtado

Dias Filgueira Thé¹, Francisco Filipe Carvalho da Silva¹, Maria Teresa Jansem de Almeida Catanho

1

Departament of Clinical Dentistry,School of Pharmacy, Dentistry and Nursing, Federal University of Ceará, Fortaleza -CE-Brazil.

2

Departament of Physiology and Pharmacology, School of Medicine. Federal University of Ceará, Fortaleza -CE-Brazil.

3

Pharmacy and Psychology Graduation Courses,Catholic Faculty Queen of Sertão, Quixadá -CE-Brazil.

4

Federal University of Pernambuco, Recife-PE-Brazil.

Corresponding author

Edilson Martins Rodrigues Neto

Jorge Acurcio Street 600, 206,

CEP 60.410-802, Fortaleza-Ce, Brazil. +55 85 32724867; +55 85 999385790 edilsonmrneto@hotmail.com

Copyright © 2016 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ARTICLE INFO ABSTRACT

Article history

Received 17/02/2016 Available online 31/03/2016

Keywords

Laser Therapy, Low-Level;

Magnetic Field Therapy; Technetium;

Wound Healing.

The energy density of low power, laser and electromagnetic field, has been used in recent years quite often in the medical field. The study of labeling red blood cells with technetium 99m is used for various evaluations in nuclear medicine including the study of blood volume in neurological intensive care. The aim was to evaluate the influence of non-ionizing radiation on the red blood cells labeled in vitro. Blood samples from rats of the Wistar strain divided into two groups were used. The first group used the EDTA and heparin as anticoagulants and received low-power laser induction (LPL) in the following separately energy density: 3, 6, 9 and 18J/cm². The second group received an electromagnetic field (EMF) of 60 Hz for 2, 4, 17 and 21 hours separately. After the induction of the laser and the electromagnetic field, the blood samples were labeled with technetium-99m (99mTc). The results indicate that the presence of anticoagulants is able to modify the uptake of technetium-99m by red blood cells. The laser induced decreases the binding capacity of the technetium-99m from 3J/cm². From the foregoing it was found that the radiation is non-ionizing EMF with LBP or alters the binding capacity of 99mTc the erythrocytes and the use of heparin as an anticoagulant gave also a higher labeling efficiency compared with the use of EDTA.The non-ionizing radiation has a promising power applications of biomedical sciences can be used for marking targets macromolecules in the body in order to diagnostic procedures and disease control.

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INTRODUCTION

Science has evolved rapidly bringing every day a new laboratory and clinical application technology. The low power laser (LBP) has been used in several areas: Physics, Biology, and Health in the various specialties [1]. The LBP has been mainly applied as an aid in tissue repair, pain relief and control of inflammation and edema. It is increasingly common the use of LBP apparatus for therapeutic purposes, especially in surgical area [2].

Science and the technology used in the treatment and diagnosis of certain diseases have been increasingly related to nuclear medicine. This is based on use of radiation for therapeutic and diagnostic purposes. In the 60s, technetium 99m (99mTc), an artificial radionuclide was introduced in medical practice. Other radionuclides also used for this purpose and scientific research are: Cobalt-57, phosphorus-32, indium-111, iodine-123, iodine-125, iodine-131, mercury-203, gold-198 and thallium-201 [3].

Technetium 99m is currently the most widely used radionuclide in nuclear medicine for diagnostic procedures and scientific research [3]. This radionuclide has advantages that facilitate its use, for example, by easily obtaining 99Mo/99Te portable generators that can be placed within a diagnostic unit occupying little space and of low cost; half-life of six hours; negligible corpuscular energy; small dose of radiation when used in tests; radioactive waste and negligible environmental damage, and enable the marking of different kinds of molecules and cells [3,4].

Red blood cells labeling with 99mTc was first performed by Fisher and colleagues in 1967 and is considered a good potential radiopharmaceutical for study, but had low labeling efficiency and rapid clearance [5]. Red blood cells labeling appears as a simple and effective method in clinical research in cardiac function, thanks to the discovery of using a reducing agent for technetium [6]. This technique is based on the intracellular ratio of reduced 99mTc and the beta chain of hemoglobin (Hb) the 99mTc 0-4 freely enters and leaves the red blood cells by diffusion, but in the presence of stannous ion (Sn+2) in the middle of it is reduced intracellular reacting with Hb, being approximately 75,8 + 2,3% on the globin and 19 + 1,5% linked to the theme [7,8]. Being connected to Hb 99mTc will be set within the red blood cells [9]. Among the various reducing agents normally used stannous chloride bi-hydrated (Sn Cl² 2h²O) [10]. Stannous chloride in the presence of 99mTc is reduced to the state of +5,4+ or 3+ [11].

The work aims to induce low power laser (LBP) at different energy densities (l/cm²), the electromagnetic field of 60Hz on the marking of hematic with pertechnetate through in vitro study of Wistar rat erythrocytes.

MATERIALS AND METHODS Obtaining blood samples

The study was approved by the ethics committee and the same male Wistar rats were used, with 90 days old, weighing between 200-250g and albino strain of mice - Swiss, aged 60 days, weighing 20-30g, from the animal house of the Department of Biophysics and Radiobiology, Federal University of Pernambuco. The animals received daily water and standard ration "ad libitum", artificial lighting, and dark light cycle of 12 in 12 hours and at room temperature between 24 and 26ºC.

Blood samples were obtained from mice by cardiac puncture in the presence of 10% EDTA anticoagulant for each 5ml of blood and heparin 5000Ul/ml.

Laser induction in blood samples

The laser emitting apparatus was the DMC brand with operating voltage from 70 to 240V, power of 9W, visible emission wavelength of 685nm, useful power of 35MW and a half active gallium phosphide and aluminum indium (InGa AIP).

Samples of blood cells contained in tubes in the presence of EDTA and heparin, making a total of five tubes per experiment, were subjected to laser at different energy densities 3,6, 9:18 J / cm² and the control tube was not submitted to the laser.

Induction of electromagnetic field (EMF) in samples

Blood samples for a total of five tubes per experiment were treated with EDTA and 10% heparin and subjected to an electromagnetic field 60Hz position at different times of 2, 4, 16 and 21 hours, respectively. The control blood cells were placed in a place where there was no electromagnetic field.

Method of appointment of red blood cells and plasma proteins with the Tc-99 m

After exposure to the laser and the electromagnetic field, samples of blood with anticoagulant were subjected to labeling of red blood cells and plasma proteins with sodium pertechnetate was added to each initially subjected to a vacuum test tube containing the blood 0,5 ml of stannous chloride, bi-hydrate (SnCl².2H²O) at a concentration of 1,2 ug/ml. To obtain this concentration stannous chloride was diluted in saline (0,9% NaCl) and prepared immediately before use. Technetium was obtained just before, from a Molybdenum-Technetium generator (99 Mo/99m Tc) which diluted sodium pertechnetate and put him in grass counter device to measure its radiation.

It was then placed in each tube 3,7Mbq stable technetium diluted mega incubates for 10 minutes. Subsequently the tubes were placed in a refrigerated centrifuge at 1500 rpm for 10 minutes. After this period of centrifugation, the plasma was aspirated fractions (supernatant) fraction and the cell and collected in separate tubes containing 0,9% saline.

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After completion of the separation of blood fractions, they were placed in the DPC gambyt Cr apparatus for measuring Tc uptake in each fraction. Through these percentage results were used the following formulas to obtain Tc activity percentage in plasma cells, in the soluble fraction of the cells. The formulas for determining the efficiency of labeling of blood elements with 99mTc are shown below:

Formula for calculating the percentage of activity (% ATI) in the different fractions obtained after centrifugation and precipitation with TCA 5%.

Plasma: FP/FP + FC In cells: FC/FP + FC

FIP or FIC FSP or Fsc: FI/FS + FI or FS/FS + FI

Statistical analysis

The experiments to investigate the effect of the laser and electromagnetic field to dial red blood cells and plasma proteins with 99mTc in vitro studies were performed with a number of five determinations. The results were calculated and expressed by the mathematical mean of the values obtained and the standard deviation thereof. The results were compared using the t-test of Student, p<0,05.

RESULTS

Induction of blood cells LBP with EDTA as an anticoagulant

The introduction of laser blood cells in the presence of EDTA at different energy densities varied with 3, 6, 9 and 18 J/cm². It is observed in Figure 1 that there was a reduction in red blood cells in the uptake of 99mTc in energy densities 3 and 6 J/cm² and 9 J/cm², there was an increase in the uptake of 99mTc in normal erythrocytes is 63,45 %, representing the control (induction 0).

Figure 1 - Evaluation of 99mTc uptake by total blood cells in the presence of EDTA, after laser induction (J/ cm²), Data express averages and standard deviation p = 0.005.

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Figure 2: Evaluation of the uptake of 99mTc by soluble blood cells after laser induction (J/cm²) Data express averages and standard deviations p = 0,005. There was used EDTA as anticoagulant.

Induction of blood cells with LBP using heparin as an anticoagulant

In Figure 3, it is observed that in blood cells, the presence of heparin increased the binding of capture in the order of 93% when compared to the control (induction 0), and there is a reduction in uptake on energy 3 J/ cm² significantly. This development also shows the 99m Tc-binding capacity with plasma and when precipitated with TCA 5%, the soluble fractions of cells remain largely unchanged.

Figure 3: Capture Evaluation of 99mTc by total blood cells in the presence of heparin, after laser induction (J/ cm²). The data express medium and standard deviation p = 0.005.

Induction of blood cells with 60 Hz electromagnetic field

In the irradiation study of CEM cells in the blood, in period 2, 4, 17 and 21 hours was observed that the red blood cells after the induction period when compared to the time 0 (zero) after an exposure to between 2 and 4 hours, uptake of 99mTc is reduce d significantly for p = 0,001 and p = 0,009 RBC into the plasma. Plasma is observed an increase of capture with 0,001 and then the pickup decays as time of exposure, not observed significant changes in erythrocytes, have a slight tendency to increased capitation no significant difference compared to the times 2 and 4h, as figure 4 shows.

Figure 4: Assessment of uptake of 99mTc by total red blood cells in the presence of heparin after the induction of CEM expressing the data mean and standard deviation p = 0.005 using EDTA as anticoagulant.

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DISCUSSION

The state laser induced in blood cells in vitro showed that the presence of EDTA promotes uptake of Pertechnetate reducing erythrocytes from marking 63% in energy density from 3 to 6 J/cm², while using the heparin increase in the uptake of pertechnetate 93% in control, significantly reduced from 3 J/cm² it shows that EDTA chelation as racing tin binding site, preventing the reduction that facilitates the marking of 99mTc in hemoglobin [12].

In vitro studies have demonstrated and suggested that the intracellular stages marking sequences which include the reduction process, 99m sodium pertechnetate by the foreign +2 (n + ²), generating the reduced 99m Tc-binding hemoglobin. The transport system Sn + ² and 99m for the internal compartment of the cell can involve Ca ++ channels and the transport and the band 3 anion and [10].

The results showed that LBP and EMF are able to modify the uptake of 99mTc in the presence of different energy densities. This indicates that the presence of certain non-ionizing radiation in the blood can modify the characteristics of radiopharmaceuticals, altering its biological behavior or their labeling efficiency.

In the literature there is no well-defined study model to evaluate the interaction of radiopharmaceuticals with non-ionizing radiation. Similarly, the assessment of the effects of drugs on the labeling of blood constituents with radionuclides has not been well defined experimental model [11]. However several authors report that non-ionizing radiation can interfere with the marking of red blood cells, white blood cells and plasma proteins labeled with 99mTc and the ¹¹¹In [12,13].

The study of non-ionizing radiation, such as laser and the electromagnetic field influencing the labeling of red blood cells and plasma proteins with 99mTc, is based on three main factors: The first is due to the importance of labeling of blood elements in nuclear medicine in general and in their use also in neurosciences. The second factor is the increasing number of people who are using laser therapy and are exposed to the electromagnetic field of low power in their daily lives [2].

The third factor is the action of LBP and EMC on the membrane of red blood cells promotes an indirect action in marking by induction of ion channels that could be competing with the pertechnetate entry and preventing 99mTc binding in hemoglobin of red blood cells [14].

These results corroborate the findings of this study, since EDTA reduces the 99m Tc-binding capacity. Since several studies have been conducted with the presence of extracts vegetables that are able to change or not the marking of red blood cells, increasing the knowledge of the possibility of drug interactions with radiopharmaceuticals which may cause misunderstanding in radio diagnostics.

The application of laser diode InGa AIP proved simple and safe and the machine easy to use, as described in [15] literature.

CONCLUSION

From the foregoing it was found that the radiation is non-ionizing EMF with LBP or alters the binding capacity of 99mTc the erythrocytes and the use of heparin as an anticoagulant gave also a higher labeling efficiency compared with the use of EDTA.

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