Investigation of the Effect of Extremely Low Frequency (ELF) Pulsed

Electromagnetic Field (PEMF) on Collagenase Enzyme Kinetics

Istiaque Ahmed, Vuk Vojisavljevic and Elena Pirogova

School of Electrical and Computer Engineering, Royal Melbourne Institute of Technology (RMIT) University,

368 Swanston Street, Melbourne 3001, Australia

Keywords: ELF PEMF System, Magnetic Field Intensity, Application, Collagenase Enzyme.

Abstract: In our earlier work we have discussed the design and development of an extremely low frequency (ELF)

pulsed electromagnetic field (PEMF) system that produces time varying magnetic field in the range of 0.5mt

to 2.5mT. 2D and 3D simulation results of the induced magnetic field produced by the system of two pairs

of air core Helmholtz coils were also presented. In this study we present the modified version of the ELF

PEMF system and discuss its application to study the effect of varying parametric changes of ELF PEMF

radiation on Collagenase enzymes that plays a key role in the process of wound healing. The findings from

this study can be used to determine the optimal characteristics of the applied ELF PEMF for wound

treatment.

1 INTRODUCTION

Power absorption in biological structures exposed to

electromagnetic irradiation is a basic aspect of the

study of biological effects of electromagnetic waves.

Causes of induced biological effects have often been

associated with the minimum electromagnetic (EM)

energy necessary to affect different chemical

reactions. However, electric and magnetic fields

may affect the metabolically supplied energy, which

might affect the rate of biologically important

chemical reactions (Sebastian, 2001).

There is much evidence that experimental

therapies in the vicinity of extremely low frequency

(ELF) part of the electromagnetic spectrum

(Juutilainen and Lang, 1997) have been emerging

for various medical conditions, such as non-uniform

bone fracture, skin ulcers, migraines and

degenerative nerves. In clinical practice LF PEMF

applications offer the possibility of more economical

and effective diagnostics and non-invasive therapies

for medical problems, including those considered

refractory to conventional treatments. Application of

PEMF to traumatic but non-infected injuries allows

the tissue to repair more rapidly. Accelerating the

rate of healing (Goudarzi et al., 2010) would reduce

both the likelihood and effect of secondary

complications.

Each and every biological process involves a

number of interactions between protein and their

targets. These

interactions are based on the energy

transfer between the interacting molecules. Enzymes

are proteins crucial in accelerating metabolic

reactions in the living organism. Study of biological

effects of applied electromagnetic radiation (EMR)

at molecular level is the main focus of this paper.

Here we aimed at investigating the efficacy of

ELF PEMF to modulate the bioactivity of the

Collagenase enzyme and thus to promote the rate of

wound healing. We present and discuss the modified

and improved ELF PEMF system which is applied

to study the effects of varying parametric changes of

ELF PEMF irradiation on Collagenase enzyme. We

also present the findings from the experimental

evaluation of the exposure system.

2 MATERIALS AND METHODS

The need of modification to our previously proposed

ELF PEMF system (Ahmed et al., 2012) stems from

the fact that activity of different biological samples

is temperature dependent. This criterion necessitates

the design for the new cuvette holder and that of the

custom made temperature controlled water

regulatory system.

143

Ahmed I., Vojisavljevic V. and Pirogova E..

Investigation of the Effect of Extremely Low Frequency (ELF) Pulsed Electromagnetic Field (PEMF) on Collagenase Enzyme Kinetics.

DOI: 10.5220/0004239501430147

In Proceedings of the International Conference on Biomedical Electronics and Devices (BIODEVICES-2013), pages 143-147

ISBN: 978-989-8565-34-1

 2013 SCITEPRESS (Science and Technology Publications, Lda.)

2.1 Improved Design of Cuvette Holder

The design of the previously used cuvette holder

only sufficed for biological experiments conducted

at room temperature. Conventional cuvette holders

can only be used for experimental studies that

require external irradiation but not in the ELF range

(Pirogova et al., 2008). Utilization of commercially

available cuvette holders under ELF PEMF radiation

is not recommended since the metal part of the

holder will interfere with the field required to

irradiate the biological sample placed in the cuvette.

Therefore, the need of a non-metallic cuvette holder

becomes inevitable. Figure 1 shows two designs and

compares between the previous and modified

versions of our cuvette holder.

Figure 1: Visual comparison between the previous cuvette

holder (left) with the modified cuvette holder (right).

Figure 2: A close up view of the cuvette holder clearly

showing the laser cut water channel (left) and the resulting

modified ELF PEMF exposure chamber (right).

The entire cuvette holder setup is made of acrylic

glass in order to eliminate the alteration of magnetic

field produced by the system of Helmholtz coil. Four

cuvette holder stands support the entire structure of

the cuvette holder. The sliding base of the holder is

fitted through these stands and made it stable by the

slide plastic screw. This screw can easily be

manoeuvred to attain the desired vertical positioning

of the cuvette holder during experimentation. The

base plastic screw at the bottom of the cuvette holder

helps to eject the cuvette after each experiment is

conducted.

Two plastic pipes are fitted to the cuvette holder

(one at the top and the other above the sliding base).

The pipe above the sliding base is connected to the

water pump and forces temperature controlled water

inside the cuvette holder through the designed laser

cut water channel (Figure 2). The pipe at the top part

of the cuvette holder channels the water back to the

water reservoir for continuous circulation. The

modified version of the ELF PEMF chamber

comprising of the newly designed cuvette holder is

presented in Figure 2.

2.2 Integration of Custom Made Water

Regulatory System

A major challenge is to create and maintain a

specific temperature in the vicinity of the cuvette.

For this, a regulated water circulatory system needs

to be integrated in the cuvette holder. Readily

available temperature control devices cannot be used

since they are connected to the holder via connectors

with metallic pins. For better understanding of the

working methodology of the custom made water

regulatory system, a schematic design is presented in

Figure 3.

Figure 3: Schematic of the custom built water regulatory

system used as part of the ELF PEMF system.

Foam board have been used as insulation

material and were placed between the outer and

inner walls of the water reservoir. This also ensures

that the maintenance of the desired water

temperature. The connection of the water pump to

the cuvette holder has been presented in the section

2.1. The water reservoir also contains a water heater

connected to a thermostat with a digital display of

water temperatures. The temperature sensor from the

thermostat is also submerged in the water reservoir.

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The thermostat maintains the water temperature and

accordingly turns on and off the water heater. The

entire process ensures that the water temperature (in

our case, 37

◦

C) inside the reservoir is well

maintained and regulated.

However, the sample temperature (with no

magnetic field) also needed to be tested to see its

relation to that of the reservoir’s water. It was done

with the help of continuous monitoring of a digital

thermometer submerged into our sample of 0.1ml

Collagenase enzyme solution. The time for the heat

to tansfer from the water in the resrvoir to inner

surface of the holder onto the cuvette and then

finally to its contained sample solution was noted

down. For accuracy, we repeated the process three

times.

The main objective was to achieve an

approximate temperature of 37

◦

C within our sample.

We observed that for our designed structure it takes

approximately 56±2 minutes for the heat to transfer

from the water in the reservoir to the internal area of

the cuvette without the sample solution. Once the

solution is put in, it takes another 2.5±0.5 minutes

for the sample temperature to reach up to the desired

temperature of 37

◦

C. Once the temperature reaches

◦

C (the same as that of the reservoir), the

temperature remains maintained for the duration of

our experiment. Therefore, before running our series

of experiment and more specifically before

irradiating our sample with magnetic field, we make

sure that the cuvette holder is sufficiently warmed

up for the heat to transfer to our solution. This is

ensured by allowing our custom made water

regulatory system to commence operating

approximately an hour before the experiment is

conducted.

2.3 Equipment

The designed ELF PEMF system was used to

produce a three dimensional region of uniform

magnetic field intensity in the range of 0.5-2.5mT

and in the range of 2-500Hz. This field was

measured by direct measurement using “Wandel and

Golterman” EFA-200 EMF Analyzer fitted with an

external B-probe. Also used for the experiment was

Spectrometer USB2000 coupled with USB-ISS-

UV/VIS (Ocean Optics, Inc.), range 190nm-870nm,

CCD detector with 2048 pixels, USB-2 connection

with Pentium IV (Windows XP), controlled with

OOIBase32 software. Software automatically

monitors and saves the absorption coefficient at

570nm every 15sec.

2.4 Measurement of Collagenase

Enzyme Activity

2.5ml cuvettes are filled with the following

components:

1. 25mg of Collagen Type I (SIGMA) added to 5ml

of Buffer A (50mM TES Buffer with 0.36mM

Calcium Chloride in 1000ml deionized water, pH

7.4) and filtered through a 0.8µm syringe filter.

2. 0.1ml of Collagenase enzyme solution (0.075

mg/ml Collagenase in Buffer A).

Experiments were performed at a temperature of 37

◦

C. This temperature was maintained using the

custom made water regulatory system described in

the section 2.2. The cuvettes were filled with 0.10ml

of Collagenase enzyme solution. These solutions

were previously irradiated with ELF PEMF

exposure system at the selected magnetic field

intensities (0.5-2.5mT) for 10min.

Before considering a standard time of irradiation,

we conducted our experimentation with selective

frequencies over the range of 2-500Hz. We observed

that irradiating our sample wih magnetic field of 0.5-

2.5mT over a period of 10 minutes significantly

increased the value of absorbance coefficients.

However, irradiation of ELF PEMF for anything

above approximately ten minutes failed to yield any

significant changes in the value of absorbance

coeeficeint. Therefore, the time for irradiation at all

frequencies and corresponding magnetic field

intensities were fixed to 10 minutes.

The irradiated enzyme solutions were then added

to the already prepared solution of Collagen Type I

and TES Buffer. The absorption coefficients were

measured at 570nm. We repeated above experiment

twice for each selected frequency (3Hz, 5Hz and

500Hz) of the applied magnetic field intensities.

3 RESULTS AND DISCUSSION

The designed and developed exposure system can be

used to study the effects of ELF PEMF radiation on

activity of selected protein solutions involved in the

process of wound healing. In particular, results

presented in this section aids to investigate the

efficacy of ELF PEMF to modulate the bioactivity

of the Collagenase enzyme. Experiments were

conducted over the range of 2-500Hz. Selected

results are presented below. Figures 4, 6 and 8 show

the change in absorbance value for our irradiated

and non-irradiated biological sample for 3Hz, 8Hz

and 500Hz respectively. Figures 5, 7 and 9 show the

InvestigationoftheEffectofExtremelyLowFrequency(ELF)PulsedElectromagneticField(PEMF)onCollagenase

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145

relative percentage increase of the absorption

coefficient corresponding to five unique values of

magnetic field intensities for each frequency

mentioned above. Differences were considered

significant at P ≤ 0.05.

Figure 4: Changes in values of absorbance (at 570nm) in

time after irradiating with 3Hz from 0.5-2.5mT.

Figure 5: Magnetic field intensity dependent effect on

Collagenase enzyme solution at 3Hz.

Figure 6: Changes in values of absorbance (at 570nm) in

time after irradiating with 8Hz from 0.5-2.5mT.

The results showed that PEMF exposures in the

frequency range of 2-500Hz and magnetic field

intensity of 0.5-2.5mT increase the Collagenase

bioactivity that results in accelerating the overall

reaction. We can clearly observe that all values of

absorption coefficient after irradiation at 3Hz and

8Hz with the magnetic field intensity range of 0.5-

2.5mT yielded a positive increase of over 9.3% and

10% respectively. A significant increase of 30% was

recorded at the exposures of 3Hz and 2.5mT, and of

19.1% at 6Hz and 2.5mT. The findings reveal that

the overall percentage change of absorption

coefficients in the range higher than 10Hz is

significantly lower. The reason behind this is the fact

that the percentage change in almost one or two

individual values in each combination of frequency

range of 12Hz-500Hz and magnetic field intensity of

0.5-2.5mt is negligible. This is clearly evident from

Figure 9, where maximum percentage increase of

12.38% can be seen at 500Hz and 2mT. The

minimum increase of 0.12% is observed at 500Hz

and 1mT.

Figure 7: Magnetic field intensity dependent effect on

Collagenase enzyme solution at 8Hz.

Figure 8: Changes in values of absorbance (at 570nm) in

time after irradiating with 500Hz from 0.5-2.5mT.

Figure 9: Magnetic field intensity dependent effect on

Collagenase enzyme solution at 500Hz.

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Figure 10: Magnetic field intensity (1.0mT) dependent effect on Collagenase enzyme solution for selected frequencies from

2-500Hz.

Figure 10 shows the plot of percentage change of

absorption coefficient versus selected frequencies

corresponding to ELF PEMF irradiation with the

magnetic field intensity of 1.0mT.

Figure 10 illuminates the fact that the overall

percentage increase in the absorption coefficient is

indeed signficantly higer acrros the lower end of the

ELF spectrum (2-10Hz) as opposed to frequencies

above 10Hz. . In contrast, the percentage increase

within each frequency in the range of 12-500Hz

varies and is generally lower than what we find in

the extremely lower end of the ELF spectrum. One

possible reason might be the fact that magnetic field

remains switched on for longer for lower frequencies

as opposed that for higher frequencies. The

relatively longer time span of the switching on and

off of the magnetic field might create a significant

amount of electric field inside the sample and

eventually aid in the overall enhancement of

Collagenase bioactivity. In summary, the results

clearly show that the absorption coefficients for the

entire array of 2-10Hz yield significant percentage

increase.

4 CONCLUSIONS

We investigated the effects of the ELF PEMF (2-

500Hz) on bioactivity of Collagenases enzyme. Prior

to carrying out the experimental investigation, we

made the essential modification to our previously

developed ELF PEMF exposure system. The

experimental results from this study clearly confirm

the possibility that protein activity can be

influenced/modulated by the external ELF PEMF.

Findings of our investigation have direct implication

in determining the optimal characteristics of the

applied ELF PEMF for possible treatment of

medical condition, in our case, wound healing

promotion.

REFERENCES

Ahmed, I., Vojisavljevic, V., Pirogova, E., 2012. Design

and Development of Extremely Low Frequency ELF)

Pulsed Electromagnetic Field (PEMF) System for

wound healing promotion. In World Congress on

Medical Physics and Biomedical Engineering, IFMBE

Proceedings 39 pp 27-30.

Goudarzi, I., Hajizadeh, S., Salmani, M. E., Abrari, K.,

2010. Pulsed electromagnetic fields accelerate wound

healing in the skin of diabetic rats. Bioelectromagnetic

31 (4) pp 318-23.

Juutilainen, J., Lang, S., 1997. Genotoxic, Carcinogenic

and Tatragenic effects of electromagnetic fields:

Introduction and overview. Mutation Research 387:

165-171.

Pirogova, E., Cosic, I., Fang, J., Vojisavljevic, V., 2008.

Use of infrared and visible light radiation as modulator

of protein activity. Estonian Journal of Engineering

14, 2, pp 107-123.

Sebastian, J. L., Munoz, S., Sancho, M., Miranada, J. M.,

2001. Analysis of the influence of the cell geometry,

orientation and cell proximity effects on the electric

field distribution from direct RF exposure. Phys. Med.

Biol. 46 213-225.

InvestigationoftheEffectofExtremelyLowFrequency(ELF)PulsedElectromagneticField(PEMF)onCollagenase

EnzymeKinetics

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