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REPORT OF RESEARCH WORK

RADIATION MEDICINE SCIENTIFIC CENTER
of MEDICAL SCIENCES ACADEMY of UKRAINE
(RMSC MSA of Ukraine)

EXPERIMENTAL RADIOLOGY INSTITUTE (ERI)
of RMSC MSA of Ukraine

APPROVE
Director of ERI
of RMSC MSA of Ukraine

MD, Professor M.I. Rudnev
Signature
October 31, 2000

Seal of Medical Sciences Academy of Ukraine,
Radiation Medicine Scientific Center 
Identification code 34837835


REPORT
OF RESEARCH WORK
to study radioprotective properties of
ß-carotene solution made of carrot in vegetable oil Karatelka


ENTRY

 

It is paradoxical that on the edge of millennia, in time of rapid development of new technologies it is more often among all age categories of population of developed countries of the world the deficit of many vitamins, mineral elements, unsaturated fat acids, different food fibers, biologically active components of plant and animal tissues is revealed [1].

In Ukraine this problem is deeper. In later years among the population of our country the massive deficit of almost all vitamins and many minerals is observed [1].

Besides, the consequences of Chernobyl catastrophe, which led to greater deterioration of the already difficult technogenic and ecological situation in many regions of Ukraine, also have negative influence on the health of the population. So, the population, which lives on radiation contaminated territories, has the increased quantity of disorders of endocrinal, nerve and immune systems activity. Cytogenetic disorders are observed, the quantity of diseases of the respiratory system, gastro enteric tract and others has increased.

The analysis of endocrine complications of population, which suffered in result of catastrophe in Chernobyl Nuclear Power Station (ChNPS), showed, that the fastest and strongest pathological response is obtained from thyroid, as overwhelming majority of radioactive emissions during the catastrophe formed the radionuclides of iodine, which have the high organotropy to thyroid [2].

The frequency of occurring of saccharine diabetes and obesity has increased. Prolonging of psychoemotional influence, which has formed the condition of chronic stress, supported the high secretion of adrenocorticotropic hormone, cortisole, insulin and other hormones, and this played an essential role in increasing of diseases of cardiovascular system (hypertensive illness, progressive atherosclerosis).
While researching the hyperphysical-gonadic system condition rather appropriate changes are revealed. Men, in particular with existence of vegetative and sexual disorders, had the decline of androgenic and generative functions of testicles; the level of testosterone was falling [3].

Increasing of autoimmune endocrine diseases frequency autoimmune thyroiditis, thyrotoxicosis, saccharine diabetes, some forms of hypocorticism is potentiated by regular disorders of immune system, changes of cellular and humoral immunity.

Regulation of all metabolic processes in living organism is done with the help of interaction of nerve, endocrine and immune systems and different fluctuations of these systems rates have one purpose the preservation of homeostasis.

That is why the protection of human organism from the different factors of environment influence, including radiation, is one of the actual problems of the present. In order to decrease the pathologic influence of unfavorable ecological factors a lot of protective means exist. So, the usage of classical radioprotectors, which were developed for the protection from the irradiation in high doses, in these conditions is not effective, as they have side effects and can be used only during the short period of time. In connection with this, the particular attention now is paid to the usage of natural, non-toxic means, which can be used during long time.

Food stuffs is very important source of vital energy of a human being, the base of the development and supporting of his physical condition, one of the most important factor of his mental activity.

It was found that the organism of a human being for its optimal functioning must receive about 600 substances or as they also called nutrients every day. They include aminoacids, vitamins, microelements and macroelements, organic acids, food fibers and others [4].

During the last time while forming food stuffs the attention is often paid to immunemodulating, radioprotecting, antistressing and other features, which food stuffs are to have, i.e. they have to be physiologically functional. Because they contain micronutrients and macronutrients, and certain functional purpose inhering in them.

Micronutrients (vitamins and vitally necessary mineral matters) belong to essential factors of human feeding, without applying of which in certain quantities valuable physiological existence of man as a living being is impossible, namely: metabolism of organism, its normal functioning, and resistance of organism to illnesses and unfavorable factors of environment.

Among biologically active food components, which influence on the health of man, an important role belongs to carotenoids. Carotenoids are one of the basic food sources of vitamin A, deficit of which in organism courses the violation of growth, reproduction processes, nerve system activity, decreasing of organism resistance to different infectious diseases [5].

Carotenoids, and ß-carotene first of all (it is almost 2 times more active than α- and γ-forms), draw attention of researchers in connection with their antioxidation and immunestimulating activity. It is known, that ß-carotene belongs to lypophile substences, is absorbed in an intestine, and this process is activated by fats and is slowed by pectin and some cholesterol decreasing means (cholestyramine) or fat substitutes (polyethers of saccharose). Differences of revealed in different oils carotene absorbing can in some degree depend on content of unsaturated fat acids and tocopherol, which are present in these oils. Possibility of man to ß-carotene assimilation is limited and in usual conditions only 9-17 % is absorbed. Intensity of ß-carotene transformation is defined by feed conditions and general condition of organism. The largest influence of alimentary factors has quality and quantity of food albumen and its aminoacid content. Albumen insufficiency decreases transformation of ß-carotene into vitamin A. Absorption and assimilation of ß-carotene depends also on kind of basic sources of ß-carotene, method of their culinary treatment and food content. Homogenization and grinding of vegetable products, their cooking and making puree of them adding butter considerably increase ß-carotene absorption.

Form intestine ß-carotene is transported into liver, where it is preserved for a long period of time. When it is necessary it is transported from liver into blood, where ß-carotene circulates mainly of 75-80 % as complexes with lipoproteins of low density [7]. Besides liver, ß-carotene as unchangeable can be deposited in a range of other organs. Effect of carotenoids also considerably conditioned by their ability to build in membrane phospholipido-albumen structures, stabilizing them and unsaturated fat acids, which are included into content of membranes [7].

It is known, that ß-carotene, being a trap for singlet oxygen, easily react with peroxide radicals. Thanks to the system of unsaturated double connections it has considerable reactive ability concerning light, oxygen, warmth and so on. As other carotenoids it has high nonspecific reactive ability and tropicity to free radicals, which are formed with influence of negative factors of environment. Thanks to presence in content of its molecule of two ionic rings ß-carotene realizes provitamin function [7].

Thanks to ability of ß-carotene to neutralize connections with free radicals it is used as antioxidant in treatment of a range of ecodepending and other pathologies.

There are known radioprotective properties of ß-carotene, in the basis of which there are immunestimulating features [8]. The use of ß-carotene stimulates the immune system processes. It stimulates proliferation of B- and T-lymphocytes, which play a very important role in defining of immunity condition with acting of free radicals, which appear during radioactive irradiation and acting of other unfavorable ecological factors. Unlike vitamin A, ß-carotene is not a poison in large quantities, though the pigmentation of hands skin can be changed, which disappears if the use of ß-carotene stops [9]. It has been probed, that taking of high doses of ß-carotene (30 mg/day) during a short period of time stimulates immune system in the people with immunity disorders and together with it does not course excessive stimulation in healthy persons [7].

It has been probed, that high doses of ß-carotene (60 mg/day) can increase immune function even in people with AIDS [10].

It is known, that oxidation of lipoproteins of low density is considered to be an initial link in development of atherosclerosis. Thanks to its antioxidant properties ß-carotene and other carotenoids are able to prevent this and protect organism from forming of atherosclerotic plaques, preventing so from cardiovascular diseases. Researches showed that ß-carotene influences on cholesterol level in irradiated animals. Under influence of a diet with ß-carotene it was revealed the true decreasing of cholesterol quantity and monoglycerides in kernels of timocytes of control and irradiated rats [11].

It was found an explicit dependence between the quality of ß-carotene usage and death rate because of cardiovascular diseases. Death risk in that group of people, which used rich in ß-carotene products, was 2 times smaller [12].

ß-carotene also normalizes processes of oxidation phosphorilyzation in liver tissues of irradiates rats [13, 14].
It was probed an anticarcinogenic activity of ß-carotene. High levels of carotenoids in food were connected with decrease of risks of some kinds of cancer forming [15].

So, the acting of ß-carotene, as it is known, is very wide and is not limited only by its factor of transmission into vitamin A, but also connected with its high antioxidant activity. It blocks free radical oxidation, coursed by radiation and toxic chemical substances, shows radioprotective properties, decreases risk of oncologic diseases forming, intensifies metabolism, increases resistance to infectious illnesses, improves visual acuity and so on. It is very important in present conditions, as consequences of Chernobyl catastrophe are felt till now, in 14 years after accident, and in addition every year industrial enterprises and transport emit in atmosphere almost 6 million tones of harmful substances 150 kg per inhabitant of Ukraine [16].

In this situation permanent sources of carotene are necessary, which will provide its necessary quantity in day ration of adults and children.

So, the analysis of literary sources permits to recommend the usage of ß-carotene with prophylactic purpose for contingents, which forms the risk groups people, who live in unfavorable ecological zones, and also for prophylaxis of cardiovascular, oncologic and other diseases and just as healthy food stuff.

For the organism normal functioning it is necessary to receive from 3 to 5 mg of carotene every day [17].

The basic raw source of carotene in our country is carrot and products of its processing.

 

Materials and research methods

 There were examined radioprotective properties of ß-carotene solution made of carrot in vegetable oil Karatelka at the liquidators of Chernobyl disaster suffered from lymphopenia (25 males, at the age of 40-60 years old) being under a regular medical check-up in the Central Military Hospital of the Ministry of Defense of Ukraine. The greater part of people being examined suffered form radiation influence received in a dose of 25 sGy.

Control group consisted of apparently healthy people (11 males, at the age of 40-60 years old) who passed annual preclinical examinations on the Hospital basis.

Examination is aimed at a study of biochemical, hormonal and immunological indices of the liquidators of Chernobyl disaster with lymphopenia upon a course of administration of ß-carotene solution made of carrot in vegetable oil Karatelka.

ß-carotene solution made of carrot in vegetable oil Karatelka oil TU U (Terms of Reference of Ukraine) 23721535.002-98, agreed by the Ministry of Health of Ukraine on 26.12.97 (Conclusion of the Ministry of Health of Ukraine under the No. 5.08.07/3427) is produced by VITAGRO CJSC (Kyiv region, Brovary).

Organoleptic indicators of Karotelka: dark-orange color, indiscrete mass, transparent oily liquid without odor.

Sunflower oil taste without off-taste. Organoleptic indicators meet the requirements set.

As to the physical-chemical indicators Karotelka meets the requirements set. Contents of heavy metals, arsenic, pesticides, nitrates, aflatoxin B1 and zearalenon do not exceed the permissible rates set by Medical-biological requirements and sanitary standards of quality of alimentary raw materials and food products. Microbiological indicators of Karotelka meet the requirements set by sanitary-technical control of preserves production at a place of production.

ß-carotene solution made of carrot in vegetable oil Karotelka contains vitamins complex: provitamin A - β-carotene (20 mg), vitamins E (50 mg) and K (28 mg) per 100 g, as well as coenzyme Q10 [ 5 ]. Energy value of 100 g of Karotelka makes up 899 calories.

Karotelka was prescribed in amount of 2 dessert-spoonfuls (at 5 mg of β-carotene per day), once a day 20-30 minutes before meal within 30 days [ 5 ].

Biochemical indicators were determined in plasma and blood erythrocytes. Blood was taken from the examined ones in the morning and fasting. Blood plasma was received by means of an established method. Chemical luminescence of blood plasma was carried out at a quantum-chemical unit 1-01, applying hydrogen hydroxide as an initiator of peroxidation. General parameters of the chemical luminescence were calculated: h height of a fast burst (in c. u.), characterizing a content of lipids hydroperoxides in a sample, S total photo-sum (in c. u.), characterizing a number of peroxy radicals appeared within a development dynamics of free-radical reaction in a medium [ 18 ]. Chemical luminescence coefficient was calculated as S ratio to h [ 19 ].

There were defined catalase activity [ 20 ], diene conjugates [ 21 ], erythrocytes acid resistance [ 19 ], general content of cholesterine under standard set of Lachema (Czech Republic).

There were also defined content of thyroid hormones thyreotrophin (TT), triiodothyronine (T3), thyroxine (FT4) and of adrenal hormone cortisole in a blood serum of the liquidators of Chernobyl disaster suffered from lymphopenia. Serum was received by means of blood centrifuging under 3000 rpm within 10 minutes. Hormones in the blood serum were defined by radioimmunologic method [ 22, 23 ].

Levels of immunoglobulins AgA, IgG, IgM in the examined persons blood were also examined.

Statistical processing of the data obtained was carried out by means of statistic analysis methods [ 24 ].

 

Results of research

In order to define the radioprotective properties of Karatelka a group of people was selected who took part in the liquidation of consequences of Chernobyl disaster and had changes in their immune status. During analyses of the immune indicators results it was defined that the liquidators of that group had stable lymphopenia in 14 years after the disaster.

We carried out the researches of Karotelka effect within a process of activation of lipids peroxidation (LPO) in the liquidators with lymphopenia. It is known that the products of radiation interacting with the biological tissues are active forms of oxygen radicals and peroxides and that the reaction of these very active products induces a chain of biological processes which realize a process of radiolesion, up to a death of a living system. Thus, examination of a process of LPO gives information which reflects a content of processes, caused by radiation influence.

During examination of the indicators of a state of free radical processes in the selected group of people in comparison with the control one, there were noticed damages of in a system of blood LPO. Thus, in the control group the rate of catalase activity didnt exceed 15 nm/min x ml, meanwhile in the group of liquidators with lymphopenia this index was statistically accurately increased and made up 22 nm/min x ml. Upon taking ß-carotene solution made of carrot in vegetable oil Karatelka within 30 days the liquidators with lymphopenia demonstrated statistically accurate decrease of catalase activity from 22 1 to 7,6 1,3 (P<0,05) and of diene conjugates from 1,57 0,25 to 0,72 0,14 (P<0,05) in comparison with these indicators before Karatelka administration (Table 1).

Table 1. Karotelka effect on indicators of the state of free radical processes and cholesterol in a blood of the liquidators with lymphopenia (Mm).

Indicators
For Karotelka taking, n = 25
After 30 days of Karotelka taking, n = 25
Catalase (nm/min x ml) 22 1 7,6 1,3*
Diene conjugates (c. u.) 1,57 0,25 0,72 0,14*
Total cholesterol (mm/l) 6,2 0,4 6,2 0,2

Notes: * - P<0, 05 statistically accurate changes related to these indicators before Karotelka taking.

 

Numerous researches proved that LPO activation is a characteristic component, necessary constituent, mechanism of stress reaction of an organism not only to radiation but other extreme agents as well. Retardation of stress-reaction may be achieved by means of preventive administration of antioxidants.

In our research a decrease of catalase activity and diene conjugates to normal indicators upon Karotelka course points at Karatelka having antioxidant properties and thus protecting a liquidators organism with lymphopenia from free radicals lesion.

An influence of ß-carotene solution made of carrot in vegetable oil Karotelka on indicators of chemical luminescence of the blood serum of the liquidators with lymphopenia was examined (Table 2).

Table 2. Karotelka effect on indicators of chemical luminescence of the blood serum of the liquidators with lymphopenia (Mm).

Indicators of blood serum chemical luminescence For Karatelka taking, n = 25
After 30 days of Karatelka taking, n = 25
Total photo-sum (c. u.) 464511 39818 149343 27782*
Height of fast burst (c. u.) 2845 279 823 142*
Coefficient of chemical luminescence 166,7 7,4 177,5 4,8

Notes: * - P<0, 05 statistically accurate changes related to these indicators before Karotelka taking.

 

It was noticed statistically accurate decrease of the total photo-sum and height of fast burst in liquidators with lymphopenia upon Karotelka taking related to these indicators for this food supplement taking.

There was examined effect of Karatelka on sensitivity of erythrocytes membrane of liquidators with lymphopenia.

A number of researches demonstrated that a system of biological membranes is one of the critical structures being a target for ionizing radiation effect, as well as other negative factors. In the first place, this concerns the external plasma membrane of a cell, complicated poly-functional, lipoproteide structure, which perceives and transfers information (almost any exo- and endogenous influence) inside a cell, regulates vital activity thereof [ 25 ]. Erythrocyte is a classical example of memranology. And erythrocytes persistence is an indicator which integrates the properties of the plasma membrane structure, cytoplasm and physiological state and depends on many reasons: erythogenesis quality, age, endo- and exogenous effects, which erythrocyte passes through within a life thereof.

Liquidators with lymphopenia after Karatelka taking within 30 days demonstrated statistically accurate increase of erythrocytes persistence to these indicators (time of hemolysis beginning,  time of maximal hemolysis and duration thereof) before Karotelka taking (Table 3).

Table 3. Karotelka effect on acid resistance of erythrocytes of the liquidators with lymphopenia (Mm).

Indicators of acid resistance of erythrocytes Before Karotelka taking,
n = 25
After 30 days of Karotelka taking, n = 25
t min (sec) 132,3 5,4 220,9 6,4*
t max (sec) 226,4 10,4 317,7 10,7*
Duration (sec) 365,5 21,3 430,9 12,5*

Notes: * - P<0, 05 statistically accurate changes related to these indicators before Karotelka taking.

 

Thus, its possible to state that ß-carotene solution made of carrot in vegetable oil Karatelka has membrane stabilizing properties and may be used as a membrane stabilizing remedy for taking by the population residing in radiation contaminated regions.

In 14 years after Chernobyl disaster we may observe a great number of thyroid lesion diseases (hypothyroidism, innocent and malignant growths, lymphocytic thyroadenitis). Also after a long time there were noticed dysfunctions of adrenal bodies characterized by increase of cortisole level in blood and egestion with urine of 11-oxicorticosteroides. Such changes were found at those people who had been working for a long time within a 30-km zone (notwithstanding a total radiation dosage per working period) [ 26 ].

That is why we carried out examination of hormones level in blood serum - thyreotrophin (TT), thyroxine (free) (T4), triiodothyronine (T3) and cortisole of the liquidators with lymphopenia before and after Karotelka taking within 30 days (Table 4).

Table 4. Effect of Karatelka on hormones level in blood serum of the liquidators with lymphopenia (Mm).

Indicators of acid resistance of erythrocytes Before Karatelka taking,
n = 25
After 30 days of Karatelka taking, n = 25
Thyreotrophin (mIU/l) 2,02 0,525 1,96 0,376
Free thyroxine (nmole/l) 13,11 0,66 12,57 0,604
Triiodothyronine (nmole/l) 2,55 0,109 2,8 0,131
Cortisole (nmole/l) 616,23 68,168 342,44 43,520*

Notes: * - P<0, 05 statistically accurate changes related to these indicators before Karatelka taking.

 

Before Karatelka taking the liquidators with lymphopenia had increased level of cortisole in comparison with the control group, accordingly 616, 23 68, 168 and 424, 56 13, 57 nmole/l in the control. After Karotelka taking the liquidators demonstrated statistically accurate decrease of cortisole content to normal values from 616, 23 68, 168 to 342, 44 43, 520 nmole/l, (P<0,05 ). Indicators of thyroid hormones TT, PT4, T3 were within the limits of control values. 

Thus, statistically accurate decrease of stress hormone cortisole in the blood serum of the liquidators with lymphopenia upon administration of Karotelka proves that Karotelka has anti-stress and adaptive features and may be recommended at nervous overstressing as well as for protection of the population residing at radiation contaminated regions.

Researches were carried out as to the Karotelka effect on the level of immunoglobulin in the blood serum of the liquidators with lymphopenia upon 30 days of taking thereof (Table 5).

According to the data received there were no statistically accurate changes in immunoglobulin level of the liquidators of Chernobyl disaster with lymphopenia in comparison with the norm as well as upon Karotelka administration.

Table 5. Karotelka effect on immunoglobulin level in the blood of the liquidators with lymphopenia (M).

Indicators
Before Karotelka taking,
n = 25
After 30 days of Karotelka taking, n = 25
Ig G (g/l) 11,42 0,244 12,07 1,282
Ig A (g/l) 1,7 0,219 1,5 0,024
Ig M (g/l) 0,68 0,037 0,64 0,110

 

Upon a course of ß-carotene solution made of carrot in vegetable oil Karotelka within 30 days the liquidators with lymphopenia demonstrated a significant improvement of the general health state. The examined ones fatigability decreased, mood improved, activity appeared, ability to work increased, appetite improved as well as gastrointestinal tract work.

No examined one demonstrated side effects in a form of allergic reactions upon Karotelka taking.

 

Conclusions

In result of the carried out researches it was established that ß-carotene solution made of carrot in vegetable oil Karatelka had antioxidant, membrane-stabilizing and anti-stress properties and thus, facilitates to organism protection under conditions of ionizing radiation.

ß-carotene solution made of carrot in vegetable oil Karatelka is recommended to everybody as therapeutic food supplement to food with radioprotective properties:

  • for organism provision with beta-carotene, vitamins E, K, coenzyme Q 10;
  • for organism protection under conditions of ionizing radiation influence and other negative factors;
  • under stress and nervous overstressing conditions;
  • for normalization of metabolism in organism and decrease of a risk of oncologic diseases emergence;
  • for organism strengthening.

Daily dosage of Karatelka:
Adults 2 dessert-spoonfuls (5 mg of beta-carotene) once a day 20-30 minutes before meal or with meal (salads, first and second courses);
Children over 1 year old 2 tea-spoonfuls (1 mg of beta-carotene) once a day with meal.

Application of the therapeutic food supplement to food Karotelka with antioxidant, membrane-stabilizing, radio-protective and adaptive properties is recommended for those who suffered from Chernobyl disaster, as well as for the population resided in the radiation-contaminated and other ecologically-negative regions, for people with increased psycho-emotional state, characterized by increased content of stress hormones, as well as a therapeutic remedy for all categories of the population with a purpose of organism strengthening and prevention of any diseases.

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