All posts in Biochemiluminescence Analysis

Ozone Treatment Control

Ozone Treatment Control

A great number of investigations have shown that ozone therapy exerts an influence on the processes of blood coagulation and lipid peroxidation (LPO). In this connection in the course of ozone therapy along with routine clinical data it is necessary to analyze coagulogram and intensity of LPO.

For objective evaluation of the state of peroxidation processes it is recommended to use the methods accepted by an informative majority of authors (Afanasyev I.B., 1984; Baraboy V.N., 1993; Bilenko M.V., 1989): induced bio chemiluminescence (IBChL), determination of the level of LPO molecular products – diene and triene conjugates, malone dialdehyde (MDA) and Schiff’s bases, tissue concentration of antioxidant enzymes –  catalase, superoxidedismutase (SOD), glutathione peroxidase and glutathione reductase as well as non-enzymatic antioxidants – a-tocopherol, ceruloplasmin, glutathione. In consideration of the fact that no one of the above parameters alone allows evaluating the level of lipid peroxidation and antioxidant activity to the full extent, it is recommended if possible to use the complex of methods.

Induced bio chemiluminescence

Bio Chemiluminescence analysis is considered a high-sensitive, economical, fast and informative method (Burlakova E.B., 1975). It allows to determinate the intensity of lipid peroxidation processes and activity of antioxidant system in biological substrates. It can be numbered to express-methods. It is enough simple and very convenient for the evaluation of lipid peroxidation in dynamics in the course of treatment.

The method of chemiluminescence induced by hydrogen peroxides and ferric sulfate (Kuzmina E.I., Nelubin A.S., Schennikova M.K., 1983) is based on that in the given system a catalytic decomposition of hydrogen peroxide occurs by metallic ions of alternating valency – two-valent iron:

ROOH + Fe2+ ® Roo. + OH + Fe3+ (Fenton’s reaction)

The resulted free radicals Roo. and OH react an activation of free-radical oxidation in biological substrate that leads to formation of instable tetroxide which decomposition is associated with emission of light quantum registered on the biochemiluminometer. The intensity of luminescence process is influenced by the whole complex of compounds producing both the oxidant and prooxidant action (Vladimirov Yu.A., Scherstnev M.P., 1989).

The biochemiluminescence methods are not direct methods of quantative determination of radical concentration, but registrate light quanta produced in reactions with active forms of oxygen (Vladimirov Yu.A., 1996). In the process of light quanta formation there are a few sequential stages:

1)     chemical conversions of initial reagents into excited molecules – biochemiluminescence emitters;

2)     transformation of excited state;

3)     decomposition of excited product associated with light quantum (Fig. 1).

Biochemical reactions

Active forms of oxygen

Electron excited products

Light quanta

Fig. 1. Formation process of light quanta in reactions with active forms of oxygen

The Nizhny Novgorod Research Center “Bioautomatic” has developed the biochemiluminometer linked to computer in dialogue mode. The device is in permanent process of improvement. The biochemiluminometer consists of:

  • cuvette section for the sample to be investigated, its thermostabilization and mixing, photoregister, amplifier of analog and counting modes, power source;
  • measuring block as a specialized micro IBM that ensures registration of signal intensity and its light sum within the given time, constant of increase and decrease in BChL signal (Ermolin S.V. et al., 1990).

We recommend using the methods developed by E.I. Kuzmina et al. (1983).

  1. Evaluation of peroxide resistance of erythrocytes by means of BChL method.

Centrifuge blood for 15 min at 2000 rotations/min, after that separate plasma from erythrocyte mass. For analysis take 1 ml of erythrocyte mass. Wash the erythrocytes twice with physiological saline. For this purpose pour into the centrifuge tube 1 ml of erythrocyte mass and 9 ml of physiological saline. Centrifuge the mixture at 2000 rotations/min for 10 min, after that pour out physiological saline. Add to the washed erythrocytes physiological saline to 5 ml.

Reagents: 0,05 mM ferric sulfate solution (weight 0,14 g FeSO4 dissolves in 50 ml of distilled water), 2% hydrogen peroxide solution (2 ml of standard perhydrol dissolves in 28 ml of distilled water), phosphate buffer (weights KH2PO4 1,361 g and KCI 3,9144 g per 0,5 l of distilled water, adjust the required pH by means of concentrated solution KHO (pH = 7,5)).

Add into the graduated cuvette 0,4 ml of ferric sulfate solution, 0,1 ml of erythrocyte mass, 0,4 ml of phosphate buffer and 0,1 ml of fresh-prepared hydrogen peroxide solution. Immediately place the cuvette on the measuring position of chemiluminometer and measure light emission within 30 sec.

  1. BChL analysis in blood plasma

The reagents are the same.

Add onto the graduated cuvette 0,2 ml of ferric sulfate solution, 0,2 ml of plasma, 0,4 ml of phosphate buffer and 0,2 ml of fresh-prepared hydrogen peroxide solution. Immediately place the cuvette with the prepared mixture on the measuring position of chemiluminometer and measure light emission within 30 sec.

The values of BChL are demonstrated in the form of kinetic curve (Fig. 2). For its evaluation the most informative parameters are used:

1)    I max (impulse/sec) – maximum luminescence intensity reflects the potential capacity of biological object for LPO; its value depends on the concentration of total lipids (TL) in blood plasma, therefore additionally calculate the correlation Imax/TL;

2)    S – light sum (within 30 sec) in plasma primarily reflects the content of radicals Roo. corresponding to breakdown of free-radical oxidation chain, inversely proportional to the activity of antioxidant system (Vladimirov Yu.A., Artchakov A.I., 1972); for determination of antioxidant activity calculate the correlation S/TL;

3)    Correlation I max/S characterizing the total antioxidant activity of blood plasma (Kontorschikova K.N., 1995);

4)    tga – the parameter characterizing the remission rate of free-radical processes in plasma and correlates with index Imax/S;

5)    the main parameter reflecting the peroxide resistance of erythrocytes is light sum (S) of BChL signal. It characterizes physiological stability and functional activity of erythrocyte membrane.

I max (impulse/sec)

time (sec)Ozone Treatment Control

Fig. 2. Typical kinetic curve of BChL signal

Evaluation of blood coagulation system

In the course of ozone therapy, especially by using parenteral methods, it is necessary to investigate the coagulation system of blood, as ozone introduced by parenteral route decreases blood coagulation. For this purpose the simple methods of determination of blood coagulation time can be used. If your clinic has a possibility to use the complex methods, the special attention should be devoted to the following parameters:

  • activated partial thromboplastin time,
  • prothrombin index,
  • thrombin time.

These are the parameters, which quickly change during intravenous ozone therapy.

Share Button