**4. Discussion**

The results of our studies suggest the existence of significant changes in the lipid metabolism, as well as sex and age differences in the lipid and lipoprotein metabolism both in healthy animals and in animals with MS.

In male hamsters fed with a high-calorie diet atherogenic dyslipidemia develops independently of age (Table 1). As it can be seen from the data obtained, increase of the total lipid content in the animal blood plasma is caused by increasing of the ApoB-containing lipoprotein (ApoB-LP) level since the HDL content is not changed. At the same time it has been found that the plasma TAG level in young (47%) and in adult animals (30%) increased in comparison with the intact group.

Increase of the TAG blood content in conditions of MS is considered to be a key factor for development of atherogenic dyslipidemia that is typical for this pathology [30]. A strong correlation between hypertriacylglycerolemia plus the HDL-C level decrease and accumulation of LDL*B* in the blood plasma has been demonstrated in many experiments and clinical studies [18].

It is assumed that atherogenic alterations occur as a result of lipoprotein disbalance in the blood plasma, i.e. because of predominance of the LDL and VLDL fractions over the


antiatherogenic HDL fraction (especially when the values of the LDL+VLDL/HDL index are higher than 3.5).

The data presented as mean±SD

\* –р0.05 versus intact animals

452 Lipoproteins – Role in Health and Diseases

The plasma cholesterol ester transfer protein (CETP) activity was examined using the modifications of Khosla et al. [44]. The CETP activity in duplicate10-μL aliquots of the plasma was determined after incubations with 3H-cholesterol ester (CE)-labeled HDL3 and LDL. Radioactivity transferred from 3H-HDL3 to LDL (measured in the supernatant after precipitation with heparin/MnCl2+) was used to calculate the CETP activity (expressed as the

To measure endothelium-bound LPL, the perfusion solution was changed to buffer containing 1% fatty acid–free BSA and heparin (5 units/ml). The coronary effluent was collected in timed fractions over 10 min and assayed for the LPL activity by measuring the

The plasma LCAT activity was measured by determination of the amount of radioactivity in each spot calculating the free cholesterol/ total cholesterol ratio in each plasma sample before and after the LCAT reaction and thus estimating the esterification rate [28]. The fractional esterification rate *(%* . h') expressed as the percentage of the free cholesterol

The HL activity was evaluated using the glycerol-stabilized emulsion of triolein and egg phosphatidylcholine containing glycerol-tri[9,10(n)-3H] oleate by determination of the

**Statistical analysis**. All data were analyzed for statistical significance with SPSS 13.0 software. The data were presented as means ± standard deviation. Statistical analysis used

The results of our studies suggest the existence of significant changes in the lipid metabolism, as well as sex and age differences in the lipid and lipoprotein metabolism both

In male hamsters fed with a high-calorie diet atherogenic dyslipidemia develops independently of age (Table 1). As it can be seen from the data obtained, increase of the total lipid content in the animal blood plasma is caused by increasing of the ApoB-containing lipoprotein (ApoB-LP) level since the HDL content is not changed. At the same time it has been found that the plasma TAG level in young (47%) and in adult animals (30%) increased

Increase of the TAG blood content in conditions of MS is considered to be a key factor for development of atherogenic dyslipidemia that is typical for this pathology [30]. A strong correlation between hypertriacylglycerolemia plus the HDL-C level decrease and accumulation of LDL*B* in the blood plasma has been demonstrated in many experiments

It is assumed that atherogenic alterations occur as a result of lipoprotein disbalance in the blood plasma, i.e. because of predominance of the LDL and VLDL fractions over the

one-way ANOVA. P<0.05 was considered to be statistically significant.

percentage of radioactivity transferred from 3H-HDL3 to LDL per 16 h of incubation).

hydrolysis of a sonicated [3H]triolein substrate emulsion [27].

esterified in the plasma sample per hour.

radioactivity amount during incubation [29].

in healthy animals and in animals with MS.

in comparison with the intact group.

and clinical studies [18].

**4. Discussion** 

**Table 1.** Some plasma lipid values in male Syrian golden hamsters with MS (in each group n=10).

As it is known, there are 2 phenotypes of LDL: LDL*A* and LDL*B* that differ by size, density, the lipid content and the atherogencity coefficient. LDL*B* have less size (d 25.5-25.75) comparing to LDL*A* (d > 25.75) and are characterized by a lower content of polar lipids, as well as a higher content of cholesterol esters. Lipoproteins of this subfraction are slowly removed from the bloodstream that is caused by their low affinity to B/E-receptors for LDL, higher sensitivity to glycosylation and oxidative damage [31]; they also have a high affinity to scavenger-receptors of macrophages [32].

All this features explain a high atherogenicity of LDL*B* subfraction. Numerous clinical and epidemiological studies have confirmed that accumulation of LDL*B* in the blood is an independent risk factor for atherosclerosis occurrence [33].

Normally, there are predominantly LDL*A* in the blood plasma, and LDL*B* are present in a small percent of the total LDL, but in MS and insulin resistance the LDL*B* content increases significantly.

It is well-known that in MS the key factor for TAG and ApoB-LP accumulation in the blood is the VLDL hyperproduction by the liver [34]. According to our data, accumulation of ApoB-LP in the blood occurs parallelly with increase in the content of this lipoprotein fraction in the liver (Table 2).

These results allow us to make a suggestion that VLDL formation is activated in the liver of the animals fed with a high-calorie diet in our experiment.

The mechanisms of the VLDL hyperproduction by the liver in the conditions of FFA intensive supply to hepatocytes have remained still unclear. The stimulation of VLDL formation can occur both by using the elevated uptake of the blood FFA and via activation of fatty acid biosynthesis *de novo* because of hyperglycemia.


The data presented as mean±SD

\* –р0.05 versus intact animals

**Table 2.** Some liver lipid metabolism values in male Syrian golden hamsters with MS used in the current study (in the crude tissue, in each group n=10).

It is known that in insulin resistance FFA that come to hepatocytes from the blood are primarily used for the TAG re-synthesis. It leads to increase in the intracellular TAG content and correlates with the increase of the VLDL secretion rate into the bloodstream. The VLDL morphology, which is specified predominantly at the second stage of their formation, depends significantly on the intracellular TAG content and hepatocyte sensitivity to insulin [35]. More active phospholipase D-dependent pre-VLDL lipidation takes place in the elevated intercellular TAG content and insulin resistance of hepatocytes [36]. Insulin blocks the VLDL1 formation in the liver. In the conditions of insulin resistance this effect and the elevated intercellular TAG content stimulate formation and secretion predominantly of VLDL1 by the liver [37].

The VLDL1 secretion increase leads to significant changes in the lipid and lipoprotein metabolism in the blood: the increased TAG content and accumulation of LDL*B* with high atherogenicity in the blood. These changes are typical for MS and considered to be separate risk factors for development of atherosclerosis.

Metabolism of ApoB-LP in the blood plasma is tightly connected with metabolism of HDL performing a reverse cholesterol transport from peripheral tissues to the liver. The leading factors in the process of transformation of VLDL into LDL in the bloodstream and determination of the LDL morphology are the rate of cholesterol esters transfer from HDL to ApoB-LP mediated by cholesteryl ester transfer protein (CETP), and the rate of TAG hydrolysis in the ApoB-LP composition mediated by lipoprotein lipase (LPL) and hepatic lipase (HL) [38].

According to data of many clinical studies, increase of the CETP activity of the HDL composition in most cases leads to decrease of the HDL-C level and accumulation of LDL*B* in the blood plasma. Moreover, a degree of these modifications correlates with the blood TAG level.

We observed significant changes in the cholesterol and HDL metabolism in the blood plasma in animals fed with a high-calorie diet. These changes have expressed the proatherogenic character and could be one of the causes for the LDL*B* accumulation in the blood.


Our results suggest that increase of the total blood cholesterol level in hamsters fed with a high-calorie diet is obviously connected with increase of the cholesterol content in the ApoB-LP composition as its level in the HDL composition decreases (Table 3).

The data presented as mean±SD

454 Lipoproteins – Role in Health and Diseases

Total lipids, mg/g liver

current study (in the crude tissue, in each group n=10).

risk factors for development of atherosclerosis.

Parameters

HDL, mg/g liver

G6PDH, nmol/mg protein/min

Lysosomal lipase, nmol/mg protein/min

ApoB-LP, mg/g liver

Week 4 Intact 104.24±2.52 11.46±0.37 1.25±0.14 3.74±0.33 0.67±0.03

Week 20 Intact 112.62±2.66 13.03±0.50 0.94±0.10 4.44±0.28 0.54±0.03

**Table 2.** Some liver lipid metabolism values in male Syrian golden hamsters with MS used in the

It is known that in insulin resistance FFA that come to hepatocytes from the blood are primarily used for the TAG re-synthesis. It leads to increase in the intracellular TAG content and correlates with the increase of the VLDL secretion rate into the bloodstream. The VLDL morphology, which is specified predominantly at the second stage of their formation, depends significantly on the intracellular TAG content and hepatocyte sensitivity to insulin [35]. More active phospholipase D-dependent pre-VLDL lipidation takes place in the elevated intercellular TAG content and insulin resistance of hepatocytes [36]. Insulin blocks the VLDL1 formation in the liver. In the conditions of insulin resistance this effect and the elevated intercellular TAG content stimulate formation and secretion predominantly of

The VLDL1 secretion increase leads to significant changes in the lipid and lipoprotein metabolism in the blood: the increased TAG content and accumulation of LDL*B* with high atherogenicity in the blood. These changes are typical for MS and considered to be separate

Metabolism of ApoB-LP in the blood plasma is tightly connected with metabolism of HDL performing a reverse cholesterol transport from peripheral tissues to the liver. The leading factors in the process of transformation of VLDL into LDL in the bloodstream and determination of the LDL morphology are the rate of cholesterol esters transfer from HDL to ApoB-LP mediated by cholesteryl ester transfer protein (CETP), and the rate of TAG hydrolysis in the ApoB-LP composition mediated by lipoprotein lipase (LPL) and hepatic

According to data of many clinical studies, increase of the CETP activity of the HDL composition in most cases leads to decrease of the HDL-C level and accumulation of LDL*B* in the blood plasma. Moreover, a degree of these modifications correlates with the blood

We observed significant changes in the cholesterol and HDL metabolism in the blood plasma in animals fed with a high-calorie diet. These changes have expressed the proatherogenic

character and could be one of the causes for the LDL*B* accumulation in the blood.

MS 124.16±2.05\* 15.16±0.54\* 1.11±0.07 2.80±0.17\* 1.09±0.07\*

MS 143.59±2.65\* 15.69±0.36\* 1.10±0.20 3.13±0.28\* 1.27±0.09\*

Age Group

The data presented as mean±SD \* –р0.05 versus intact animals

VLDL1 by the liver [37].

lipase (HL) [38].

TAG level.

\* –р0.05 versus intact animals, а –р0.05 versus intact animals 4 weeks.

**Table 3.** Plasma HDL-C and HDL-CE, cholesterol esterifying activity and CE transfer in Syrian golden hamsters with the experimental MS (in each group n=10).

Decrease in the HDL cholesterol level is apparently connected with increase of the transfer rate of cholesteryl esters from HDL to ApoB-LP. According to our data the rate of the cholesteryl esters transfer from HDL in the animals fed with a high-calorie diet grows to 166% and 199% compare to the values of young and adult intact animals, respectively (Table 3).

At the same time decrease in the free cholesterol and HDL esterified cholesterol levels was determined in young males, but in adult animals only the HDL esterified cholesterol content lowered. The cholesteryl ester transfer rate from HDL to ApoB-LP is activated when the TAG content increases in the blood, it is observed in the postprandial period, as well as in ApoB-LP metabolism abnormalities [39]. In both cases the cholesteryl ester transfer activation is a consequence of increasing the TAG-rich lipoproteins (TRL) in the bloodstream [40]. The latter is also confirmed by our data pertaining to the increase of the neutral lipids content in the ApoB-containing lipoproteins in hamsters with the experimental MS. These differences seem to be connected with the difference in the HDL free cholesterol esterification rate in males of various ages. This rate is primarily determined by the activity of LCAT – the enzyme associated with HDL [41].

The increase of the cholesteryl-ester transfer activity from HDL is mostly the result of the CETP activation. The increase of the CETP activity in MS was demonstrated in a great number of experiments [22]. It is known that the activation of CETP biosynthesis in the liver is primarily the cause for increasing the activity of this protein in the blood HDL composition, but mechanisms of CETP induction have been still unclear.

Thus, increase of the cholesteryl ester transfer rate from HDL on the background of hypertriacylglycerolemia, which is observed in our experiment in the animals fed with a high-calorie diet (Table 3), is atherogenic since the cholesteryl ester transfer predominantly to TAG-enriched lipoproteins leads to accumulation of CE-enriched VLDL1, which are major precursors of LDL*B*. Intensive TAG supply to HDL in exchange for cholesteryl esters results in accumulation of TAG-enriched HDL particles, which are the predominant

substrate for hepatic lipase (HL), in the blood. So, HDL particles are rapidly removed from the bloodstream and it leads to decrease of the HDL-C content.

That is why changes in the enzymes activity, which hydrolyze lipoprotein lipids in the bloodstream, in particular – in LPL and HL activity, affect significantly the lipoprotein metabolism in MS.

TAG in the TAG-enriched lipoproteins (chylomicrons and VLDL) are the substrate for LPL. FFA, released after hydrolysis under the action of LPL, come to adipocytes and muscle cells where they are deposited as the TAG component or used as a source of energy. TAG hydrolysis in the VLDL composition increases availability of cholesterol for its transfer to HDL, therefore, in this way LPL mediates the reverse cholesterol transfer. The LPL activity is regulated by the influence on transcription, translation and enzyme transport from the cells. Insulin is known to activate LPL that results in decrease of the total blood TAG level and stimulation of cholesterol reverse transfer [35].

According to our data, the plasma LPL activity decreased in young male hamsters fed with a high-calorie diet (Table 4).


The data presented as mean±SD

\* –р0.05 versus intact animals

**Table 4.** Postheparin plasma lipase activities in Syrian golden hamsters with the experimental MS (in each group n=10).

The results obtained are in agreement with the literature data about the reduction of the LPL activity in obesity and insulin resistance [42]. The mechanisms of the LPL activity inhibition in these conditions are still unclear though a definite contribution could be made by development of insulin resistance.

The increase of the cholesteryl ester transfer rate from HDL on the background of hypertriacylglycerolemia, which was stated in our experiment both in animals fed with a high-calorie diet and in chronic stress, is an atherogenic factor for two reasons. Firstly, the cholesteryl ester transfer predominantly to the TAG-enriched lipoprotein fractions leads to accumulation of VLDL1 enriched with cholesteryl esters, which are the main LDL*B* precursors. Secondly, the intensive exchange of cholesteryl esters in HDL for TAGs results in accumulation of TAG-enriched HDL in the blood, which are the predominant substrates for HL, and they are rapidly removed from the bloodstream, and it, in turn, causes decrease in the HDL-C concentration. The activation of the lipoprotein secretion by the liver is also observed in the conditions of the acute chemical and emotional painful stress. This fact may be considered to be a sign of proatherogenesis since it is accompanied by hyperlipidemia development due to increase of atherogenic lipoprotein fractions.

456 Lipoproteins – Role in Health and Diseases

metabolism in MS.

a high-calorie diet (Table 4).

Age (at the beginning of

The data presented as mean±SD \* –р0.05 versus intact animals

development of insulin resistance.

each group n=10).

substrate for hepatic lipase (HL), in the blood. So, HDL particles are rapidly removed from

That is why changes in the enzymes activity, which hydrolyze lipoprotein lipids in the bloodstream, in particular – in LPL and HL activity, affect significantly the lipoprotein

TAG in the TAG-enriched lipoproteins (chylomicrons and VLDL) are the substrate for LPL. FFA, released after hydrolysis under the action of LPL, come to adipocytes and muscle cells where they are deposited as the TAG component or used as a source of energy. TAG hydrolysis in the VLDL composition increases availability of cholesterol for its transfer to HDL, therefore, in this way LPL mediates the reverse cholesterol transfer. The LPL activity is regulated by the influence on transcription, translation and enzyme transport from the cells. Insulin is known to activate LPL that results in decrease of the total blood TAG level

According to our data, the plasma LPL activity decreased in young male hamsters fed with

Week 4 Intact 8±2 51±4

Week 20 Intact 83±2 3±1

**Table 4.** Postheparin plasma lipase activities in Syrian golden hamsters with the experimental MS (in

The results obtained are in agreement with the literature data about the reduction of the LPL activity in obesity and insulin resistance [42]. The mechanisms of the LPL activity inhibition in these conditions are still unclear though a definite contribution could be made by

The increase of the cholesteryl ester transfer rate from HDL on the background of hypertriacylglycerolemia, which was stated in our experiment both in animals fed with a high-calorie diet and in chronic stress, is an atherogenic factor for two reasons. Firstly, the cholesteryl ester transfer predominantly to the TAG-enriched lipoprotein fractions leads to accumulation of VLDL1 enriched with cholesteryl esters, which are the main LDL*B* precursors. Secondly, the intensive exchange of cholesteryl esters in HDL for TAGs results in accumulation of TAG-enriched HDL in the blood, which are the predominant substrates for HL, and they are rapidly removed from the bloodstream, and it, in turn, causes decrease in the HDL-C concentration. The activation of the lipoprotein secretion by the liver is also observed in the conditions of the acute chemical and emotional painful stress. This fact may

LPL (U/ml) HL (U/ml)

MS 4±1\* 91±3\*

MS 129±3\* 2±1

the experiment) Group Parameters

the bloodstream and it leads to decrease of the HDL-C content.

and stimulation of cholesterol reverse transfer [35].

As shown in our studies, decrease of the LPL activity in the blood plasma of young males fed with a high-calorie diet can be an additional factor for TAG accumulation in the blood and the HDL-C level reduction observed in our experiment.

HL mediates a selective transport of VLDL remnants to hepatocytes via LDL-receptors, takes part in reverse transport of cholesterol accelerating HDL coming into the liver via scavenger receptors (SRB1). Hydrolyzing TAG in the ApoB-LP composition HL plays a significant role in their re-modelling in the bloodstream. It is known that the HL activity specifies substantially the lipid composition, size and properties of LDL [43].

The HL activity is predominantly regulated at the transcriptional level under the influence of sex hormones, glucocorticoids and adipokines. The rate of the HL gene transcription is also dependent on the intercellular lipid content, primarily cholesterol in the hepatocytes [44].

In our experiment the blood plasma HL activity in male hamster fed with a high-calorie diet increased irrespective of age (Table 4), it corresponds to literature data. In a number of studies it has been shown that the HL activity increases in insulin resistance, obesity, and a highcalorie diet [45]. Moreover, it has been determined that increase of the HL mRNA content is observed when using a high-calorie diet; this is the evidence of the enzyme biosynthesis activation under these conditions. The authors associate this fact with the decrease in the blood plasma adiponectin level, which can inhibit the HL synthesis in hepatocytes.

Considering these data, as well as the data obtained in our studies about adiponectin decrease in the blood plasma in obesity (Fig. 1), we may suppose that one of the causes for the HL activity increasing when taking a high-calorie diet in our experiment is decrease of adiponectin secretion by the adipose tissue.

The HL activity increase is considered to be one of the key factors for the atherogenic dyslipidemia development in obesity and MS. In a number of works a clear correlation between the HL activity and the LDL*B* content in the blood plasma was demonstrated [19]. It is believed that namely HL activation results in the increased LDL*B* formation [33]. The latter occurs with increase of the TAG-enriched VLDL1 content in the blood and the CETP activation. Furthermore, the HL activity increase leads to decrease of the HDL cholesterol level [46]. This is associated with the fact that hydrolysis of TAG in the HDL3 composition results in their transformation into HDL2, which are rapidly removed from the bloodstream by the liver. Thus, the HDL-C level decrease that we determined in our experiment (Table 3) can be a consequence of the HL activity increase.

In the current study we have found that the blood FFA level increase is accompanied by the ApoB-LP synthesis activation in the liver of Syrian male hamsters fed with a high-calorie diet irrespective of age. This causes increase of the TAG and ApoB-LP level in the blood. Decrease of the HDL-C level is a consequence of the rate of cholesteryl ester exchange between HDL and LDL due to activation of CETP and HL. As a result of these changes the atherogenic dyslipidemia development, which is typical for MS, is observed.

**Figure 1.** Plasma adiponectine level in Syrian golden hamsters with the experimental MS development (values are mean±SD; \* –р0.05 versus intact animals, in each group n=10, \* – p < 0.05 versus intact animals).

We have found the age differences of the lipid profile in the blood plasma of the normal male hamsters. So, in intact males (with age from 4 to 20 weeks) on the background of the constant content of total lipids and lipoproteins in the blood plasma there was increase of the FFA level (60% comparing to a 4-week intact), TAG (48%) and ApoB-LP (20%), and the HDL level showed a tendency to decrease. These results are the evidence of the lipidation increase with age. It has been also shown that in adult males the unesterified cholesterol and cholesteryl ester levels are lower than in young animals (20% and 25%, respectively), and the cholesteryl ester transfer rate from HDL in adult animals exceeds this index value in young animals (191%) (Table 3).

The data obtained correspond to the literature data about age-dependent changes in the lipid metabolism in males, which have the proatherogenic character [47]. It is known that with age the sex hormones level lowers in males and the glucocorticoid secretion level increases. The plasma lipid profile in males is determined, among other factors, by the secretion level of sex hormones possessing antiatherogenic properties. A lot of studies proved the presence of direct correlation between the blood testosterone plus the dehydrotestosterone level and the HDL-C content [48]. Moreover, the high level of sex hormones correlates with decrease of the TAG content and the total cholesterol in the blood. Thus, increase of the TAG level and decrease of the HDL cholesterol content in the blood plasma of males with age may be connected with reduction of the sex hormone secretion (Table 5). Changes of the lipid profile in the blood plasma of males with age may be also associated with increase of glucocorticoid secretion, which was observed in our experiment (Table 5).

Thus, with age the blood plasma lipid profile in males is subjected to unfavourable changes such as increase of the FFA and TAG content and decrease of HDL-C. The latter may be connected with decrease of the sex hormone level, and increase of the cortisol secretion. However, despite the more favourable lipid profile in the blood plasma of young males comparing with normal adult animals, atherogenic dyslipidemia in obesity and insulin resistance develops irrespective of age.

In contrast with males, in females the atherogenic dyslipidemia development is significantly dependent on age (Table 6). In particular, while in males with age there are no significant changes in the liver ApoB-LP content, in females this index raises during maturation – in intact animals – to 20%, and in animals in the experimental MS – to 31%. That indicates intensification of lipolytic processes in the liver of females during ageing, and may serve as a manifestation of the lipid metabolism activation. The analogous changes in the total lipid content also proved this tendency (Table 6).


The data presented as mean±SD

\* –р0.05 versus intact animals

458 Lipoproteins – Role in Health and Diseases

young animals (191%) (Table 3).

which was observed in our experiment (Table 5).

**Figure 1.** Plasma adiponectine level in Syrian golden hamsters with the experimental MS development (values are mean±SD; \* –р0.05 versus intact animals, in each group n=10, \* – p < 0.05 versus intact animals).

We have found the age differences of the lipid profile in the blood plasma of the normal male hamsters. So, in intact males (with age from 4 to 20 weeks) on the background of the constant content of total lipids and lipoproteins in the blood plasma there was increase of the FFA level (60% comparing to a 4-week intact), TAG (48%) and ApoB-LP (20%), and the HDL level showed a tendency to decrease. These results are the evidence of the lipidation increase with age. It has been also shown that in adult males the unesterified cholesterol and cholesteryl ester levels are lower than in young animals (20% and 25%, respectively), and the cholesteryl ester transfer rate from HDL in adult animals exceeds this index value in

The data obtained correspond to the literature data about age-dependent changes in the lipid metabolism in males, which have the proatherogenic character [47]. It is known that with age the sex hormones level lowers in males and the glucocorticoid secretion level increases. The plasma lipid profile in males is determined, among other factors, by the secretion level of sex hormones possessing antiatherogenic properties. A lot of studies proved the presence of direct correlation between the blood testosterone plus the dehydrotestosterone level and the HDL-C content [48]. Moreover, the high level of sex hormones correlates with decrease of the TAG content and the total cholesterol in the blood. Thus, increase of the TAG level and decrease of the HDL cholesterol content in the blood plasma of males with age may be connected with reduction of the sex hormone secretion (Table 5). Changes of the lipid profile in the blood plasma of males with age may be also associated with increase of glucocorticoid secretion,

Thus, with age the blood plasma lipid profile in males is subjected to unfavourable changes such as increase of the FFA and TAG content and decrease of HDL-C. The latter may be connected with decrease of the sex hormone level, and increase of the cortisol secretion. **Table 5.** Plasma sex hormones and cortisol levels in hamsters with the experimental MS (in each group n=16)


The data presented as mean±SD

\* –р0.05 versus intact animals

**Table 6.** Lipid metabolism parameters in the liver homogenate in Syrian golden female hamsters with MS (in the crude tissue, in each group n=16)

Oxidation of LDL and VLDL (i.e. ApoB-LP) is an alternative way of the lipoprotein catabolism, which leads to their uptake by macrophages via scavenger-receptors, and may

lead to the transformation of these cells into "foam" ones. That is why it is one of the factors of atherogenesis in MS.

In our experiment we also observed the composition changes in lipoproteins and in particular HDL particle enrichment with lipids (Table 7). However, the cholesterol content of these lipoproteins decreased in contrast to the ApoB-LP cholesterol content that was increased.


The data presented as mean±SD or percentage

\* –р0.05 versus intact animals

**Table 7.** The plasma HDL composition in Syrian golden hamsters (1 year) with the experimental MS (in each group n=10).

There are several possible reasons for that phenomenon. One of them is a well-known fact that HDL contains high levels of both unsaturated fatty acids, which are rapidly utilized, and proteins, which hydrophilic properties compensate the lack of phospholipids, as well as α-tocopherol and enzymatic antioxidants, particularly paraoxonase, which protect these lipoproteins from peroxidation. There is no doubt that the changes in the cholesterol metabolism enzymes activity associated with HDL (CETP) are involved in this process (Table 3).

Nevertheless, the content decrease of compounds with isolated double bonds and accumulation of the lipoperoxidation products has been determined in the HDL fraction in MS (Table 7). Moreover, the data obtained have shown that the content of ketodienes and coupled trienes in the HDL fraction is 129% comparing to control; the content of diene conjugates – 168% and the content of the total hydroperoxides – 115%. It has been also found that there is decrease of the α-tocopherol content in HDL (41%) comparing to the control values (Table 7).

Thus, HDL can protect LDL from oxidation "providing" a cell with paraoxonase and PAFacetyl hydrolase. However, this protective effect of HDL is reduced in response to induction of the stress acute phase in animal models [49].

As can be seen from our data (Table 8), the HDL-associated paraoxonase activity is generally decreased in experimental MS.

Antioxidant Complexes and Lipoprotein Metabolism – Experience of Grape Extracts Application Under Metabolic Syndrome and Neurogenic Stress 461


The data presented as mean±SD

460 Lipoproteins – Role in Health and Diseases

The data presented as mean±SD or percentage

\* –р0.05 versus intact animals

control values (Table 7).

decreased in experimental MS.

of the stress acute phase in animal models [49].

(in each group n=10).

(Table 3).

of atherogenesis in MS.

lead to the transformation of these cells into "foam" ones. That is why it is one of the factors

In our experiment we also observed the composition changes in lipoproteins and in particular HDL particle enrichment with lipids (Table 7). However, the cholesterol content of these lipoproteins decreased in contrast to the ApoB-LP cholesterol content that was increased.

Parameters Group


Total lipids, % of the total HDL composition 49.451.35 57.311.91\* Total cholesterol, % of the total HDL composition 14.970.23 11.210.76\* TAG, % % of the total HDL composition 1.750.07 3.080.15\*

**Table 7.** The plasma HDL composition in Syrian golden hamsters (1 year) with the experimental MS

There are several possible reasons for that phenomenon. One of them is a well-known fact that HDL contains high levels of both unsaturated fatty acids, which are rapidly utilized, and proteins, which hydrophilic properties compensate the lack of phospholipids, as well as α-tocopherol and enzymatic antioxidants, particularly paraoxonase, which protect these lipoproteins from peroxidation. There is no doubt that the changes in the cholesterol metabolism enzymes activity associated with HDL (CETP) are involved in this process

Nevertheless, the content decrease of compounds with isolated double bonds and accumulation of the lipoperoxidation products has been determined in the HDL fraction in MS (Table 7). Moreover, the data obtained have shown that the content of ketodienes and coupled trienes in the HDL fraction is 129% comparing to control; the content of diene conjugates – 168% and the content of the total hydroperoxides – 115%. It has been also found that there is decrease of the α-tocopherol content in HDL (41%) comparing to the

Thus, HDL can protect LDL from oxidation "providing" a cell with paraoxonase and PAFacetyl hydrolase. However, this protective effect of HDL is reduced in response to induction

As can be seen from our data (Table 8), the HDL-associated paraoxonase activity is generally

Intact MS

\* –р0.05 versus intact animals

**Table 8.** The plasma HDL paraoxonase activity in Syrian golden hamsters with experimental MS (in each group n=10).

The data obtained show that application of antioxidant complexes for correction of unfavourable changes in proatherogenic states may be perspective since free radical oxidation activation is a common pathogenetic link of all those states; this link is not only involved in damage of cells and their components, but also as an alternative way of catabolism it accelerates the lipid recyclization.

At the same time, since a significant feature of proatherogenic states is the hormone status imbalance, polyphenolic antioxidants need special attention because these compounds along with the antioxidative activity also demonstrate phytoestrogen properties [50], and it may be an additional factor of the lipid metabolism regulation.

An important effect of flavonoids is scavenging of oxygen-derived free radicals. The experimental systems *in vitro* have also shown that flavonoids possess anti-inflammatory, antiallergic, antiviral, and anticarcinogenic properties. The so-called ''Mediterranean diet'' is thought to prevent cardiovascular diseases, as a consequence of its high content of antioxidants, which are crucial in ameliorating oxidative events implicated in many diseases. In addition to the antioxidant/antiradical activity, red wine polyphenols (RWPs) have been shown to possess many biological properties, including inhibition of platelet aggregation, the vasorelaxing activity, modulation of the lipid metabolism, and inhibition of the low-density lipoprotein oxidation.

In our research we have used wine, juice and polyphenolic extracts from grapes of different grades, and polyphenolic concentrates "Enoant" and "Polyphen" obtained from *Vitis Vinifera* grapes to correct the changes in the lipid metabolism in the conditions of the experimental metabolic syndrome, acute and chronical stress. All substances used in our research were developed in National Institute for Vine and Wine "Magarach" (Yalta, Ukraine). The studies carried out have specified that polyphenolic extracts and concentrates are quite active remedies that decrease negative effects in MS though the effectiveness of various substances administered are significantly different.

So, administration of any of the investigated substances has significantly decreased the total blood plasma lipoprotein content in hamsters with MS, but the use of "Cabernet" extract has the most pronounced effect (Fig. 2). The same tendency is observed in decreasing the ApoB-LP content, the total cholesterol and FFA level have also decreased, though practically no difference between the grape varieties investigated has found.

\* – р0.05 versus intact animals.

**Figure 2.** The effect of *Vitis Vinifera* substances on some plasma lipid metabolism values in male Syrian golden hamsters (1 year old) with the experimental MS (in each group n= 7)

The non-enzyme antioxidant level (α-tocopherol, reduced glutathione and ascorbic acid) in the blood serum has also reached reference values under the influence of the polyphenolic extracts. This fact confirms the high antioxidant activity of the studied substances.

Normalization of the blood plasma phospholipid content under the influence of polyphenolic extracts arouses the interest. The phospholipid content returned to the intact level, which may be a result of their oxidation reduction, given that the unsaturated fatty acids in phospholipids are compounds that undergo oxidation by free radicals quickly and easily.

However, in spite of the quite favourable effect of "Isabella" extract, its administration also has negative consequences, particularly the HDL level decrease to the value observed in intact animals accompanied by the LDL content increase.

The investigated substances normalize also the blood lipoproteins composition. Thus, the total lipid and the total cholesterol content decrease in the ApoB-LP composition, moreover "Enoant" lowers the cholesterol content in this atherogenic lipoprotein fraction even below the control level.

Generally, the TAG content is also normalized under the action of all the investigated substances, but taking into account the ratio – cholesterol/triacylglycerols, "Polyphen" has the most favourable effect.

The polyphenol extracts and concentrates have significantly improved the ApoB-LP oxidative status in animals with MS. The best results have been obtained when using "Cabernet", as well as for other indexes investigated (Table 9).


The data presented as mean±SD or percentage

462 Lipoproteins – Role in Health and Diseases

\* \* \* \* \*

\*

\*

\* – р0.05 versus intact animals.

0

20

40

60

80

100

120

140

160

**% of intact**

180

So, administration of any of the investigated substances has significantly decreased the total blood plasma lipoprotein content in hamsters with MS, but the use of "Cabernet" extract has the most pronounced effect (Fig. 2). The same tendency is observed in decreasing the ApoB-LP content, the total cholesterol and FFA level have also decreased, though practically no

\*

\* \* \*

\*

\*

\* \* \*

\* \* \*

**Figure 2.** The effect of *Vitis Vinifera* substances on some plasma lipid metabolism values in male Syrian

TLP АpоВ-LP TC ТГ FFA PhL AscA HDL TL

MS MS+Еn MS+Pp MS+Isab MS+Cab

The non-enzyme antioxidant level (α-tocopherol, reduced glutathione and ascorbic acid) in the blood serum has also reached reference values under the influence of the polyphenolic

Normalization of the blood plasma phospholipid content under the influence of polyphenolic extracts arouses the interest. The phospholipid content returned to the intact level, which may be a result of their oxidation reduction, given that the unsaturated fatty acids in phospholipids

However, in spite of the quite favourable effect of "Isabella" extract, its administration also has negative consequences, particularly the HDL level decrease to the value observed in

The investigated substances normalize also the blood lipoproteins composition. Thus, the total lipid and the total cholesterol content decrease in the ApoB-LP composition, moreover

extracts. This fact confirms the high antioxidant activity of the studied substances.

are compounds that undergo oxidation by free radicals quickly and easily.

intact animals accompanied by the LDL content increase.

golden hamsters (1 year old) with the experimental MS (in each group n= 7)

difference between the grape varieties investigated has found.

\* \*\* \*\*

\* – р0.05 versus intact animals, \*\* – р0.05 versus model of MS

**Table 9.** The effect of *Vitis Vinifera* substances on the plasma ApoB-LP composition in male Syrian golden hamsters (1 year old) with the experimental MS (in each group n= 10)

The HDL composition in the blood is also affected by the substances studied. In these particles the total lipid content decreases and even reaches the level of intact animals when using "Cabernet" extract (Table 10).

The cholesterol level also changes: it decreases when using "Enoant" and increases under the action of "Isabella" and "Cabernet" extracts.

The HDL-C content decreases under the action of "Enoant" may occur due to peroxide processes inhibition since cholesterol accumulation in lipoprotein particles, as it was mentioned before, has a compensatory character in response to the phospholipid oxidation of the lipoprotein particle hydrophilic cover. The TAG content decreased under the action of

all substances, and "Isabella" was the most effective substance. The TAG content decrease is probably mediated by the phytoestrogenic action of polyphenols directed to lipolysis inhibition in the adipose tissue.


The data presented as mean±SD or percentage

\* – р0.05 versus intact animals, \*\* – р0.05 versus model of MS

**Table 10.** The effect of *Vitis Vinifera* substances on the plasma HDL composition in male Syrian golden hamsters (1 year old) with the experimental MS (in each group n= 10)

The exact bimolecular mechanisms for this cardioprotection are unclear, but it is likely that actions mediated both through the estrogen receptors, such as the beneficial alteration in lipid profiles and upregulation of the low-density lipoprotein (LDL) receptor, and independently of the estrogen receptors, such as antioxidant action, contribute to the cardioprotective effects of phytoestrogens observed.

The potential role of phytoestrogens, including isoflavonoids, as cardioprotective agents has been extensively reviewed. The data obtained in our experiments showed that in male hamsters with the experimental MS the treatment with grape extracts reduced VLDL cholesterol (VLDL-C) and TG by 30 and 40 % compared with the control animals. Furthermore, golden Syrian hamsters fed with red wine phenolics had a significant decrease in the plasma apo B concentrations. Similar to our previous study, grape polyphenols may have altered hepatic secretion of TG-rich VLDL. This reduction is evident when observing the decreases in both plasma apo B and apo E concentrations. The significant decrease in apo E concentrations may have further reduced plasma TG concentrations. In general, apo E displaces apo C-II from the VLDL particle, thereby inhibiting the lipoprotein lipase (LPL) activity and overall lipolysis. Furthermore, Huang et al. [51] showed that adding apo C-II to transgenic apo-E3–enriched VLDL increased the LPL activity in a dose-dependent manner. The reductions in apo E and TG concentrations suggest less displacement by apo E, thereby promoting the grape polyphenols activity and further reducing the TG concentrations in the plasma.

464 Lipoproteins – Role in Health and Diseases

inhibition in the adipose tissue.

Parameter

Total lipids, % of the total ApoB-LP composition

Total cholesterol, % of the total ApoB-LP composition

TAG, % of the total ApoB-LP composition

Ketodienes+conjugated trienes, U/ml

The data presented as mean±SD or percentage


Isolated double bonds, U/ml 7.31

Diene conjugates, mmol/L 31.68

Total hydroperoxides, mmol/L 78.31

\* – р0.05 versus intact animals, \*\* – р0.05 versus model of MS

cardioprotective effects of phytoestrogens observed.

hamsters (1 year old) with the experimental MS (in each group n= 10)

all substances, and "Isabella" was the most effective substance. The TAG content decrease is probably mediated by the phytoestrogenic action of polyphenols directed to lipolysis

+"Enoant"

54.91 0.21\*/\*\*

10.54 0.30\*/\*\*

2.90 0.09\*/\*\*

7.47 0.20\*/\*\*

7.67 0.08\*/\*\*

24.85 0.35\*/\*\*

1.24 0.03\*\*

75.26 0.31\*/\*\*

**Table 10.** The effect of *Vitis Vinifera* substances on the plasma HDL composition in male Syrian golden

The exact bimolecular mechanisms for this cardioprotection are unclear, but it is likely that actions mediated both through the estrogen receptors, such as the beneficial alteration in lipid profiles and upregulation of the low-density lipoprotein (LDL) receptor, and independently of the estrogen receptors, such as antioxidant action, contribute to the

The potential role of phytoestrogens, including isoflavonoids, as cardioprotective agents has been extensively reviewed. The data obtained in our experiments showed that in male hamsters with the experimental MS the treatment with grape extracts reduced VLDL cholesterol (VLDL-C) and TG by 30 and 40 % compared with the control animals. Furthermore, golden Syrian hamsters fed with red wine phenolics had a significant decrease in the plasma apo B concentrations. Similar to our previous study, grape polyphenols may have altered hepatic secretion of TG-rich VLDL. This reduction is evident when observing the decreases in both plasma apo B and apo E concentrations. The significant decrease in apo E concentrations may have further reduced plasma TG concentrations. In general, apo E displaces apo C-II from the VLDL particle, thereby inhibiting the lipoprotein lipase (LPL) activity and overall lipolysis. Furthermore, Huang et al. [51] showed that adding apo C-II to transgenic apo-E3–enriched

MS MS

57.31 1.91\*

11.21 0.76\*

3.08 0.15\*

0.35\*

0.17\*

1.65\*

1.48 0.06\*

1.33\*

Group

MS+ "Isabella"

51.74 0.74\*/\*\*

> 12.05 0.21\*

1.92 0.04\*/\*\*

7.36 0.11\*/\*\*

7.99 0.05\*/\*\*

22.55 0.34\*/\*\*

1.25 0.03\*/\*\*

74.48 0.55\*/\*\*

MS+ "Cabernet "

> 49.20 0.42\*\*

12.45 0.34\*/\*\*

1.96 0.03\*/\*\*

8.13 0.06\*\*

8.15 0.01\*/\*\*

21.88 0.23\*\*

1.54 0.47\*/\*\*

73.41 0.39\*/\*\*

MS +"Polyphen"

> 53.09 0.08\*/\*\*

> > 11.14 0.04\*

2.11 +0.12\*/\*\*

7.19 0.17\*/\*\*

7.69 0.07\*/\*\*

23.44 0.40\*/\*\*

1.32 0.03\*/\*\*

75.62 0.54\*/\*\* Due to decreases in TG concentrations, administration of "Cabernet" extract was shown to affect the overall lipoprotein metabolism. Decreased concentrations of the plasma TG altered substrate availability in the delipidation cascade, leading to the decrease observed in LDL-C concentrations. After a 3-week treatment period the grape polyphenols treatment induced a significant decrease in the cholesteryl ester transfer protein (CETP) activity as well. Such decrease in the CETP activity may be partially a result of the substantial decrease in substrate availability, including both the plasma TG and LDL-C.

It is evident that grape polyphenols modify the packaging of VLDL through alteration in the hepatic enzyme activity and apo B secretion. These modifications seem to decrease the overall secretion of the VLDL particles and therefore, decrease plasma TG and related apo concentrations. Due to decrease of the TG substrate, further modifications in the lipoprotein metabolism may occur.

The alteration in the TG metabolism may not be the single mechanism driving the hypocholesterolemic effects of grape polyphenols. When golden Syrian hamsters were treated with dealcoholized red wine, red wine, or grape juice, similar significant reductions in both TC and LDL-C concentrations were apparent in all treatment groups compared with the control [51]. Although there was a trend for decrease in TG concentrations in all treatment groups compared with the control, the differences were not significant. That study, along with others, suggests the presence of an additional mechanism by which grape polyphenols exert the cardioprotective effect. In Hep G-2 cells, dealcoholized red wine was shown to upregulate significantly the LDL receptor activity. This significant increase in activity was similar to the increase seen when Hep G-2 cells were treated with atorvastatin. Furthermore, when Hep G-2 cells were treated with increasing doses of red wine, LDL receptor mRNA abundance was significantly increased in a dose-responsive manner. The increase of the LDL receptor activity and abundance may be a result of the homeostatic intracellular cholesterol feedback loop. In general, decrease in the intracellular cholesterol will upregulate the LDL receptor expression and activity, whereas increase in the intracellular cholesterol will downregulate the receptor [48]. Grape polyphenols were shown to decrease hepatic cholesterol concentrations; therefore, the liver compensates for this deficiency by upregulating the LDL receptor and the overall decrease in the plasma LDL concentrations occurs.

One possible explanation of the anti-atherogenic activity of grape polyphenols is the wellknown HDL cholesterol-increasing effect of polyphenols in various species, including transgenic mice [52].

In our experiments it has been found that the grape extract treatment induced slight (15%) increase in HDL cholesterol concentrations is possibly related to the significant decrease in the hepatic lipase activity (Table 11). The reductions observed in both hepatic and LPL activities by grape polyphenols treatment may prevent formation of small atherogenic VLDL*B* particles and may also decrease their uptake by the LDL receptor -related protein.

In addition to increases in HDL cholesterol concentrations, grape extracts also change the size and quality of HDL particles [53]. Although the mechanisms by which polyphenols influence the metabolism of HDL particles are not clear, changes in LPL and cholesteryl ester transfer protein (CETP) may play an important role.

Polyphenols treatment in humans is associated with decrease in the CETP content correlated with the concomitant increase in HDL cholesterol concentrations [54]. Consistent with our findings, grape extracts caused a significant increase in the postheparin LPL activity and HDL cholesterol concentrations in patients with moderate hypercholesterolemia and in hamsters [39]. However, the HDL cholesterol-increasing action of polyphenols in animals (mouse, hamster and rat) without CETP in some cases [52] suggests that this effect is may be independent of the CETP activity.


\*P<0.05 versus control animals.

**Table 11.** The plasma postheparin lipases activity in male hamsters with MS (in each group n=10)

The "Cabernet" extract appeared to be the most effective substance in relation to the HDL defence from peroxidation, though the other substances revealed the same but not so high activity. They decreased the content of products (diene conjugates, ketodienes+coupled trienes, total hydroperoxides) effectively and increased – substrates (compounds with isolated double bonds) of lipoperoxidation, prevented decrease of the antioxidant level (α-tocopherol).

It should be pointed out that the level of lipid peroxidation secondary products (ketodienes+coupled trienes) decreased more effectively under the influence of "Enoant" (to intact values).

Under the action of the studied substances the lipoprotein supply to the liver also decreases, evidenced by the decrease of the ApoB-LP content in the organ. Moreover, in the composition of these lipoproteins the TAG content normalizes, and it indicates normalization of the activity of lipases catalyzing the lipoprotein metabolism in the blood (Table 12).

The liver oxidative status is also improved: the antioxidant levels almost restore, the peroxidation products content decreases, the content of compounds with isolated double bonds increases (Tables 3, 5, 9, 12).

Testing of the "Enoant" action – one of the substances studied – in female hamsters of different age with the experimental MS proved the effectiveness of the antioxidant therapy of this pathology.

So, the total lipids, TAG and FFA contents decrease in the blood plasma of those animals under the action of "Enoant" (Table 12). In addition, in adult females "Enoant" causes decrease in the ApoB-LP and total cholesterol content, and it, in turn, reduces atherogenic changes in MS.

Antioxidant Complexes and Lipoprotein Metabolism – Experience of Grape Extracts Application Under Metabolic Syndrome and Neurogenic Stress 467

Parameter Group MS MS + "Enoant" MS + "Polyphen " MS+ "Isabella" MS+ "Cabernet" Total cholesterol, % of the total ApoB-LP composition. 7.180.06\* 8.230.26\*/\*\* 8.460.05\*\* 8.760.05\*\* 9.100.13\*\* TAG, % of the total ApoB-LP composition 42.001.29\* 44.640.52\*\* 44.420.43\*\* 45.950.50\*\* 45.410.73\*\* Isolated double bonds, U/g 2.130.06\* 2.390.04\*/\*\* 2.710.03\*/\*\* 2.990.09\*\* 2.770.16\*\* Total hydroperoxides, mmol/g 101.032.00\* 90.551.54\*/\*\* 88.691.02\*/\*\* 80.460.77\*/\*\* 78.832.71\*/\*\*

The data presented as mean±SD or percentage

466 Lipoproteins – Role in Health and Diseases

independent of the CETP activity.

bonds increases (Tables 3, 5, 9, 12).

\*P<0.05 versus control animals.

intact values).

of this pathology.

changes in MS.

ester transfer protein (CETP) may play an important role.

In addition to increases in HDL cholesterol concentrations, grape extracts also change the size and quality of HDL particles [53]. Although the mechanisms by which polyphenols influence the metabolism of HDL particles are not clear, changes in LPL and cholesteryl

Polyphenols treatment in humans is associated with decrease in the CETP content correlated with the concomitant increase in HDL cholesterol concentrations [54]. Consistent with our findings, grape extracts caused a significant increase in the postheparin LPL activity and HDL cholesterol concentrations in patients with moderate hypercholesterolemia and in hamsters [39]. However, the HDL cholesterol-increasing action of polyphenols in animals (mouse, hamster and rat) without CETP in some cases [52] suggests that this effect is may be

milliunits Control (MS) (n=50) Grape extract "Cabernet" (n=50)

**Table 11.** The plasma postheparin lipases activity in male hamsters with MS (in each group n=10)

bonds) of lipoperoxidation, prevented decrease of the antioxidant level (α-tocopherol).

of lipases catalyzing the lipoprotein metabolism in the blood (Table 12).

The "Cabernet" extract appeared to be the most effective substance in relation to the HDL defence from peroxidation, though the other substances revealed the same but not so high activity. They decreased the content of products (diene conjugates, ketodienes+coupled trienes, total hydroperoxides) effectively and increased – substrates (compounds with isolated double

It should be pointed out that the level of lipid peroxidation secondary products (ketodienes+coupled trienes) decreased more effectively under the influence of "Enoant" (to

Under the action of the studied substances the lipoprotein supply to the liver also decreases, evidenced by the decrease of the ApoB-LP content in the organ. Moreover, in the composition of these lipoproteins the TAG content normalizes, and it indicates normalization of the activity

The liver oxidative status is also improved: the antioxidant levels almost restore, the peroxidation products content decreases, the content of compounds with isolated double

Testing of the "Enoant" action – one of the substances studied – in female hamsters of different age with the experimental MS proved the effectiveness of the antioxidant therapy

So, the total lipids, TAG and FFA contents decrease in the blood plasma of those animals under the action of "Enoant" (Table 12). In addition, in adult females "Enoant" causes decrease in the ApoB-LP and total cholesterol content, and it, in turn, reduces atherogenic

LPL 356.0±53.2 258.6±57.3\* Hepatic lipase 232.6±25.9 216.2±34.7

\* – р0.05 versus intact animals, \*\* – р0.05 versus the model of MS

**Table 12.** The effect of *Vitis Vinifera* substances on the liver cytosol ApoB-LP composition in male Syrian golden hamsters (1 year old) with the experimental MS (in the crude tissue, in each group n= 10)

The increase of -tocopherol (the main lipid-phase antioxidant) in the blood plasma of animals that received "Enoant" proved its antioxidant activity in our experiment (Table 13).

Furthermore, the significant decrease of the body weight was observed in hamsters that received "Enoant" along with a high-calorie diet compared to the animals on a high-calorie diet alone.

Based on these findings, we may conclude that introduction of grape polyphenolic extracts and concentrates in MS can prevent the increase of the total lipid and ApoB-LP content in the blood plasma, prevent the activation of free radical processes in the plasma lipoprotein particles, and normalize the liver lipid metabolism. The ability of the investigated substances to reduce negative consequences of MS such as atherosclerosis development has been proven.

The last suggestion is confirmed by our results concerning the aorta wall lipid composition in the experimental MS. The introduction of "Enoant" for prophylaxis and treatment reduces significantly atherogenesis manifestations in the aorta, decreasing the aorta media lipidation and the neutral lipid content (Fig. 3, 4).

Thus, from our data, we can conclude that antioxidants, particularly grape polyphenolic concentrates and extracts, which have pronounced antioxidant, phytoestrogenic and stressprotector properties, should be included into a complex therapy of MS to reduce its negative effects.

The next experiment was designed to investigate the action of grape wines and polyphenolic concentrates on development of proatherogenic effects of the emotional-painful stress. In our experiments we used purebred female rats because, as it was shown in previous studies, the acute stress response in females was more expressive than in males.


The data presented as mean±SD or percentage

\*\* – р0.05 versus the model of MS

**Table 13.** The effect of polyphenol concentrate "Enoant" on some plasma lipid metabolism values in female Syrian golden hamsters with the experimental MS (in each group n= 10)


The data presented as mean±SD or percentage

\*\* – р0.05 versus the model of MS

**Table 14.** The effect of polyphenol concentrate "Enoant" on some liver lipid metabolism values in female Syrian golden hamsters with the experimental MS (in each group n= 10)

During 21 days animals were daily given *per os* grape wines of "Cabernet" and "Rkatsiteli" grades in the doses that corresponded to 300 ml of wine for a human of 70 kg. Other animals were given alcohol in the dose corresponding to 30 ml of alcohol for a human of 70 kg, as well as polyphenolic concentrates "Enoant" and "Polyphen" in the doses of 0.05 ml/kg of the body weight. The grape wines and polyphenolic concentrates were produced by the National Institute of Grape and Wine "Magarach". Control animals were given the corresponding volume of the physiological solution.

MS 4.52

MS 7.75

The data presented as mean±SD or percentage

MS+ "Enoant"

MS+ "Enoant"

The data presented as mean±SD or percentage

\*\* – р0.05 versus the model of MS

MS+ "Enoant"

MS+ "Enoant"

\*\* – р0.05 versus the model of MS

Age Group

4 weeks

20 weeks

Total lipids, mg/ml

0.17

3.54 0.16\*\*

0.20

6.88 0.14\*\* ApoB-LP, mg/ml

4.00 0.16

3.47 0.13

3.84 0.11

3.30 0.08\*\*

female Syrian golden hamsters with the experimental MS (in each group n= 10)

Total lipids, mg/g

9,15

111.53 4.08\*\*

2.70

121.04 4.18\*\*

female Syrian golden hamsters with the experimental MS (in each group n= 10)

MS 140.75

MS 154.18

corresponding volume of the physiological solution.

Total cholesterol, mmol/L

> 2.04 0.08

> 1.88 0.06

> 2.58 0.07

2.22 0.05\*\*

**Table 13.** The effect of polyphenol concentrate "Enoant" on some plasma lipid metabolism values in

 - Tocopherol, nmol/g

> 21.01 1.47

25.31 0.34\*\*

> 19.59 0.39

26.11 1.03\*\*

**Table 14.** The effect of polyphenol concentrate "Enoant" on some liver lipid metabolism values in

During 21 days animals were daily given *per os* grape wines of "Cabernet" and "Rkatsiteli" grades in the doses that corresponded to 300 ml of wine for a human of 70 kg. Other animals were given alcohol in the dose corresponding to 30 ml of alcohol for a human of 70 kg, as well as polyphenolic concentrates "Enoant" and "Polyphen" in the doses of 0.05 ml/kg of the body weight. The grape wines and polyphenolic concentrates were produced by the National Institute of Grape and Wine "Magarach". Control animals were given the

Parameter

TAG, mg/ml

1.08 0.49

0.91 0.02\*\*

> 1.40 0.04

1.18 0.03\*\*

FFA, mmol/L

> 1.17 0.06

0.95 0.02\*\*

> 1.42 0.04

1.15 0.03\*\*

> Ascorbic acid, mkmol/g

> > 3.73 0.14

4.08 0.10

6.10 0.35

5.96 0.24

Parameters

Diene conjugates in ApoB-LP, nmol/ml

> 24.82 1.46

> 22.27 0.99

> 23.58 1.35

> 21.10 1.14

 - Tocopherol, nmol/ml

> 6.67 0.22

9.79 0.77\*\*

> 10.49 0.82

12.87 0.36\*\*

TBA active substances, nmol/g

> 1.97 0.06

> 1.74 0.09

> 1.95 0.09

> 1.73 0.09

Age Group

4 weeks

20 weeks

**Figure 3.** The lipid content in the aorta wall in male Syrian golden hamsters with the experimental MS and "Enoant" treatment (Mm, in each group n=10).

**Figure 4.** The lipid content in the aorta wall in female Syrian golden hamsters with the experimental MS and "Enoant" treatment (Mm, in each group n=10).

It was shown that all the substances investigated: polyphenolic concentrates "Enoant" and "Polyphen", 10% solution of ethanol, and grape wines "Cabernet" and "Rkatsiteli" possessed the stress-protective activity, which intensity was dependent on the substance used (Tables 15-17).

When introducing only "Enoant" and "Polyphen" to the animals these complexes did not cause any changes on the investigated indexes of the pro-oxidant and antioxidant status in the liver and it is an indication about safety of using these concentrates.

"Enoant" and "Polyphen" revealed the significant protective activity in the emotionalpainful stress. It allows to use them as stress-protective, hepatoprotective and antiatherogenic remedies.


The data presented as mean±SD

\* - р0.05 versus intact animals

\*\* - р0.05 versus stressed animals

**Table 15.** The effect of grape polyphenol complexes and grape wines on the lipid metabolism and the plasma corticosterone level in rats with the neurogenic stress (in each group n=10).

Moreover, we have found out that the stress-protective activity of grape wines is equal to the polyphenolic concentrates activity given in the similar dose.

Wines of "Cabernet" and "Rkatsiteli" grades normalized the total lipid content both in a liver homogenate and in the blood plasma in stress; in addition, TAG levels also reached the control values.

Grape wine components prevented the FFA content increase noted when introducing the solution of alcohol. This fact may prove the protective action of the components mediated by inhibition of fatty infiltration of organs.

The latter is confirmed by the absence of influence of grape wine introduction on the NADPH-generating dehydrogenases activity in the liver.

The cholesterol content decrease in the blood plasma when introducing grape wines has attracted our attention, as well as a favourable redistribution of cholesterol in the LP fractions – decrease of ApoB-containing lipoprotein level with the unchanged HDL content.

Antioxidant Complexes and Lipoprotein Metabolism –

Experience of Grape Extracts Application Under Metabolic Syndrome and Neurogenic Stress 471


The data presented as mean±SD

\* - р0.05 versus intact animals

470 Lipoproteins – Role in Health and Diseases

Stress + Enoant

> nonstr.

3.98 0.31 \*\*

0.78 0.09\*

54.90 6.92 \*/\*\*

0.89 0.08

1.89 0.26\*

5.87 1.08 \*/\*\*

str.

4.14 0.45\*

0.76 0.08 \*/\*\*

70.76 9.34\*

0.14

1.56 0.12 \*\*

5.50 0.99 \*/\*\*

by inhibition of fatty infiltration of organs.

NADPH-generating dehydrogenases activity in the liver.

Group

5.50 0.74 \*/\*\*

0.66 0.12 \*/\*\*

85.71 5.71

1.10 0.19

1.85 0.19 \*\*

15.00 1.72 \*/\*\*

Stress + ethanol

> nonstr.

5.58 0.37\*

0.91 0.05 \*/\*\*

69.10 7.37 \*\*

1.00 0.12

1.67 0.18 \*\*

38.75 5.64 \*\*

Stress + Cabernet

> nonstr.

3.46 0.43 \*\*

0.58 0.04 \*/\*\*

62.25 6.50 \*\*

1.04 0.08

1.67 0.18 \*\*

26.33 4.40 \*/\*\*

str.

3.41 0.66 \*\*

0.50 0.05

56.71 7.32\*

1.26 0.15

2.07 0.29\*

32.75 5.17 \*\*

Stress + Rkatsiteli

> nonstr.

3.57 0.54 \*\*

0.54 0.05 \*\*

80.83 8.74 \*\*

0.96 0.05

1.63 0.18 \*\*

40.60 6.38 \*\*

str.

3.43 0.15 \*\*

0.54 0.10

97.51 9.66 \*\*

1.08 0.12

1.86 0.20 \*\*

16.93 2.43 \*/\*\*

Stress + Polyphen

3.81 0.03 \*/\*\*

0.51 0.08

74.14 8.21\*

0.96 0.05

1.59 0.16 \*\*

7.10 0.80 \*/\*\*

plasma corticosterone level in rats with the neurogenic stress (in each group n=10).

the polyphenolic concentrates activity given in the similar dose.

str. non-str. str.

3.82 0.48 \*\*

0.71 0.09\*

82.93 9.48 \*\*

0.88 0.08

1.54 0.22 \*\*

29.33 8.58

**Table 15.** The effect of grape polyphenol complexes and grape wines on the lipid metabolism and the

Moreover, we have found out that the stress-protective activity of grape wines is equal to

Wines of "Cabernet" and "Rkatsiteli" grades normalized the total lipid content both in a liver homogenate and in the blood plasma in stress; in addition, TAG levels also reached the

Grape wine components prevented the FFA content increase noted when introducing the solution of alcohol. This fact may prove the protective action of the components mediated

The latter is confirmed by the absence of influence of grape wine introduction on the

The cholesterol content decrease in the blood plasma when introducing grape wines has attracted our attention, as well as a favourable redistribution of cholesterol in the LP fractions – decrease of ApoB-containing lipoprotein level with the unchanged HDL content.

Parameter

TL, mg/ml

TAG, mg/ml

Total cholesterol, mg/ml

АpоВ-LP, mg/ml

Corticosterone, nmol/l

control values.

HDL, mg/ml 0.98

The data presented as mean±SD \* - р0.05 versus intact animals \*\* - р0.05 versus stressed animals

\*\* - р0.05 versus stressed animals

**Table 16.** The effect of grape polyphenol complexes and grape wines on the plasma oxidant/antioxidant status in rats with the neurogenic stress (in each group n=10).

Paraoxonase activity was normalized in the animals given wines and the antioxidant content both in the blood plasma and in the liver was significantly higher than in the control animals.

These effects together with much lower level of ApoB-LP oxidation in the animals given grape wines prove the high antiatherogenic potential of the wines investigated.

In addition, the grape wines have revealed a rather high level of the stress-protective activity and it is indicated by a significant decrease of the corticosterone content in the blood plasma in stressed animals given wines.

Since grape wines have shown a high level of the stress-protective activity we investigated how the ratio of wine components – polyphenols and ethanol – can influence the stressprotective activity of this complex.

As was shown in our experiments "Enoant" administration even in the combination with ethanol does not reduce the stress-protective action of it, but on the contrary – it intensifies this action preventing unfavourable effects of the alcohol. At the same time the TAG and FFA level in the liver tissue of rats given ethanol together with "Enoant" decreases even when using the lowest dose investigated (0.01 ml per 100 g) (Table 18). Since the content of TAG and FFA increases apparently due to the lipogenesis activation when using ethanol, which might lead to fatty infiltration of the liver, then reduction of this process activity could protect the liver.


The data presented as mean±SD

\* - р0.05 versus intact animals

\*\* - р0.05 versus stressed animals

**Table 17.** The effect of grape polyphenol complexes and grape wines on the liver lipid metabolism in rats with the neurogenic stress, in the crude tissue (in each group n=10).

Antioxidant Complexes and Lipoprotein Metabolism –

Experience of Grape Extracts Application Under Metabolic Syndrome and Neurogenic Stress 473


The data presented as mean±SD

472 Lipoproteins – Role in Health and Diseases

protective activity of this complex.

Stress + Enoant

> nonstr.

151.84

19.86 \*\*

3.77

0.47

4.43

0.07 \*/\*\*

1.00

0.11

str.

149.17

33.14

4.65

0.78

4.58

0.06

\*\*

1.06

0.12

The data presented as mean±SD \* - р0.05 versus intact animals \*\* - р0.05 versus stressed animals

Stress + Polyphen

> nonstr.

201.93

33.71 \*\*

5.94

1.08

\*\*

3.15

0.44\*

1.26

0.08\*

str.

130.98

23.66

6.00

0.32

3.72

0.34\*

1.08

0.16

rats with the neurogenic stress, in the crude tissue (in each group n=10).

could protect the liver.

Parameter

Total lipids, mg/g

TAG,

mg/g

АpоВ-LP, mg/g

FFA,

mg/g

Since grape wines have shown a high level of the stress-protective activity we investigated how the ratio of wine components – polyphenols and ethanol – can influence the stress-

As was shown in our experiments "Enoant" administration even in the combination with ethanol does not reduce the stress-protective action of it, but on the contrary – it intensifies this action preventing unfavourable effects of the alcohol. At the same time the TAG and FFA level in the liver tissue of rats given ethanol together with "Enoant" decreases even when using the lowest dose investigated (0.01 ml per 100 g) (Table 18). Since the content of TAG and FFA increases apparently due to the lipogenesis activation when using ethanol, which might lead to fatty infiltration of the liver, then reduction of this process activity

Group

nonstr.

220.54

23.22 \*/\*\*

6.89

0.49

\*\*

4.10

0.49 \*/\*\*

1.45

0.13\*

Stress + Cabernet

> nonstr.

180.31

15.98 \*\*

3.88

1.08

3.15

0.16 \*/\*\*

1.22

0.20

str.

171.39

28.66 \*\*

4.90

1.08

3.06

0.16 \*/\*\*

1.05

0.22

Stress + Rkatsiteli

str. non-str.

190.03

18.21\*\*

4.93

0.68

3.02

0.36\*

1.40

0.31

164.93

16.93 \*\*

5.74

0.61

2.87

0.30 \*/\*\*

1.15

0.13

Stress + ethanol

str.

193.32

25.61 \*/\*\*

7.86

0.25

\*\*

5.00

0.82

1.35

0.15\*

**Table 17.** The effect of grape polyphenol complexes and grape wines on the liver lipid metabolism in

\* - р0.05 versus intact animals

\*\* - р0.05 versus stressed animals

**Table 18.** The effect of grape polyphenol complexes and grape wines on the liver tissue oxidant/antioxidant status in rats with the neurogenic stress (in the crude tissue, in each group n=10).

It should be noted that the effect of high doses of "Enoant" (0.1 and 0.15 ml/100 g) was ambiguous. On the one hand, it caused α-tocopherol accumulation in the liver that might be an indicator of their protective action, but on the other hand, it probably revealed some

prooxidative effect initiating the increase in the content of the POL final products – thiobarbituric acid-active products, and also activating ApoB-containing lipoproteins oxidation. In this case the secondary oxidative stress developed. A tendency to decrease the lipid content in the liver and to increase it in the blood plasma testifies about it.

Such effect is typical for high doses of many antioxidants capable to reveal the prooxidant action, including α-tocopherol. These data indicate the necessity of reasonable attitude to antioxidants therapy, including "Enoant".


The data presented as mean±SD

\* - р0.05 versus intact animals

**Table 19.** The effect of different doses of polyphenol concentrate "Enoant" in combination with ethanol on the plasma parameters of the stress response development in rats with the neurogenic stress (in each group n=10).

At the same time small doses of "Enoant" have a relatively low biological activity; they do not reduce negative effects of ethanol intake and do not inhibit the stress response significantly.

474 Lipoproteins – Role in Health and Diseases

Parameter

Total lipids, mg/ml

Total cholesterol, mg/ml

АpоВ-LP, mg/ml


Ascorbic acid, mkmol/L

Diene conjugates in АpоВ-LP, mkmol/L

ТBA-active products, mkmol/L

Corticosterone, nmol/L

group n=10).

The data presented as mean±SD \* - р0.05 versus intact animals

TAG, mg/ml 0.91

HDL, mg/ml 0.93

antioxidants therapy, including "Enoant".

Stress +Ethanol

> 5.89 0.08\*

> 0.07\*

0.49 0.07

0.02

1.73 0.05\*

4.11 0.34\*

33.81 1.73\*

28.11 0.34\*

2.39 0.55\*

35.25 4.27

5.69 0.06\*

0.99 0.04\*

0.46 0.05

1.00 0.06

1.84 0.05\*

4.88 0.17\*

34.04 2.73\*

28.99 0.14\*

2.01 0.30\*

24.00 3.03

prooxidative effect initiating the increase in the content of the POL final products – thiobarbituric acid-active products, and also activating ApoB-containing lipoproteins oxidation. In this case the secondary oxidative stress developed. A tendency to decrease the

Such effect is typical for high doses of many antioxidants capable to reveal the prooxidant action, including α-tocopherol. These data indicate the necessity of reasonable attitude to

> 5.30 0.05\*

> 0.73 0.04\*

0.54 0.04

1.02 0.07

1.48 0.06

5.84 0.14\*

39.04 1.60\*

29.35 0.80\*

1.48 0.16\*

28.25 4.54

**Table 19.** The effect of different doses of polyphenol concentrate "Enoant" in combination with ethanol on the plasma parameters of the stress response development in rats with the neurogenic stress (in each

Group

4.82 0.15\*

0.52 0.03

0.40 0.01\*

0.91 0.05

1.44 0.03

6.68 0.24\*

49.25 1.10\*

28.81 1.30\*

1.11 0.04

16.00 0.44\*

Stress+Ethanol+ Enoant, ml per 100 g of the body weight: 0.01 0.03 0.05 0.07 0.1 0.15

> 3.51 0.08

> 0.43 0.03

> 0.55 0.09

1.81 0.03\*

1.18 0.05

8.20 0.34\*

55.01 1.67

23.91 0.51

1.18 0.34

25.50 0.50

3.44 0.09

0.39 0.02\*

0.55 0.03

1.23 0.06\*

1.41 0.02

9.23 0.35

56.98 2.03

20.60 1.43

0.77 0.21

17.50 2.33\*

3.58 0.15

0.50 0.02

0.56 0.02

1.32 0.04\*

1.64 0.04\*

8.80 0.46

49.96 3.43

28,27 1.35\*

1.31 0.20\*

21.60 6.00

lipid content in the liver and to increase it in the blood plasma testifies about it.

Therefore, we can conclude that the most effective doses of "Enoant" are 0.05-0.07 ml/100 g of the body weight because with these doses "Enoant" has not only high stress-protective, antiatherogenic and hepatoprotective activities, but practically neutralizes negative effects of ethanol.

Thus, our results suggest that grape wines have a high stress-protective, antiatherogenic and hepatoprotective activity that is equal to grape polyphenolic non-alcoholic concentrates characteristics, and the wine components in the doses studied have prevented negative effects of ethanol. Introduction of ethanol to animals in the human equivalent dose – 0.43 ml/kg of the body weight increases their tolerance to stress, but is an unfavourable factor that could result in MS development, fatty infiltration of organs and other pathologies. The polyphenolic concentrates "Enoant" and "Polyphen" in the human equivalent dose – 0.3 ml/kg of the body weight reveal a significant stress-protective, hepatoprotective and antiatherogenic activity under the action of the emotional-painful stress. Grape wines from "Cabernet" and "Rkatsiteli" grades in the human equivalent dose – 4.3 ml/kg of the body weight also reveal a high stress-protective, antiatherogenic and hepatoprotective activity equal to grape polyphenolic non-alcoholic concentrates, and the wine components in the doses used prevented the negative effect of ethanol.

The highest activity has been shown by the combination of "Enoant" and ethanol that corresponds to the ratio of components in dry red wines, as well as the absence of significant difference in the protective effects of red and white wines, in spite of the difference in the polyphenol content [55]. Based on these results, in the second series of our experiments we decided to investigate "Cabernet" and "Rkatsiteli" wine effects on the development of stress-reaction proatherogenic consequences under the action of the emotional-painful stress in different periods of introduction.

It has been shown that "Cabernet" had a higher level of the anti-atherogenic activity than "Rkatsiteli"; in relation to the stress-protective activity the wines of these grades did not differ markedly. Such effect is likely connected with accumulation of polyphenols in the organism.

To examine the last supposition it was necessary to determine how different periods of introduction of the investigated wines influenced the stress-reaction development. We have carried out the study of wine intake influence on the development of proatherogenic consequences of the emotional and painful stress in different terms after consumption.

The data obtained in the experiments showed significant improvement of the antioxidant status both in the blood plasma and the liver tissue one day after the introduction of "Cabernet" wine (tables 20, 21).

At the same time "Rkatsiteli" wine did not reveal such activity. A similar condition persisted for 2-5 days of administration.


The data presented as mean±SD

\* - р0.05 versus intact animals

**Table 20.** The effect of prophylactic administration of grape wines of "Cabernet" (C) and "Rkatsiteli" (R) grades on the stress response development in the liver tissue in rats with the neurogenic stress in different periods of time, in the crude tissue (in each group n=10).

Antioxidant Complexes and Lipoprotein Metabolism –

Experience of Grape Extracts Application Under Metabolic Syndrome and Neurogenic Stress 477


The data presented as mean±SD

476 Lipoproteins – Role in Health and Diseases

Total lipids,

mg/g TAG, mg/g GSH,

Parameter


Tocopherol, nmol/g

Diene conjugates, nmol/g

ТBA-active products, nmol/g

mkmol/g

C+Str 94.945.65\* 3.800.11\* 2.120.17\* 18.880.79\* 14.930.37\* 1.680.10

R+Str 103.164.63\* 5.150.61\* 2.370.28\* 15.600.39\* 15.940.39\* 2.240.32

C+Str 105.175.12\* 3.580.23\* 2.510.34\* 26.042.17\* 14.130.09\* 1.070.24\*

R+Str 106.939.42\* 5.670.34\* 2.770.87\* 15.650.92\* 15.100.07\* 1.910.15

C+Str 115.3811.65\* 3.920.13\* 3.820.37\* 23.692.18\* 13.850.48\* 1.700.11

R+Str 103.285.81\* 6.470.28\* 2.470.26\* 16.890.71\* 14.440.25\* 2.290.20\*

C+Str 139.148.06\* 5.570.31\* 2.430.37\* 26.962.12 13.190.34 1.350.15

R+Str 116.663.60\* 6.880.37\* 2.430.42\* 18.641.18\* 14.220.16\* 2.060.13

C+Str 161.186.05\* 7.270.15 4.110.21 32.120.85 12.280.52 1.640.13

R+Str 122.207.07\* 6.350.45 2.330.28\* 23.082.08\* 12.930.44 1.730.04

C+Str 181.829.24 8.040.63 5.150.42 30.622.53 11.860.12 1.300.19\*

R+Str 153.995.30\* 8.410.56 3.220.48\* 26.311.26\* 11.840.48 1.290.14\*

C+Str 174.726.15 8.340.55 3.740.25 32.555.58 12.420.36 1.470.27

R+Str 164.48.03\* 8.630.47 3.250.17\* 28.282.26 11.410.22 1.450.31

C+Str 162.1612.81 7.500.43 4.610.22 38.162.06\* 10.500.52\* 1.340.06\*

R+Str 172.1310.42 7.680.69 4.420.34 41.992.42 10.270.63\* 1.240.17

**Table 20.** The effect of prophylactic administration of grape wines of "Cabernet" (C) and "Rkatsiteli" (R) grades on the stress response development in the liver tissue in rats with the neurogenic stress in

different periods of time, in the crude tissue (in each group n=10).

Periods of time

Day 1

Day 2

Day 3

Day 5

Day 8

Day 10

Day 12

Day 15

The data presented as mean±SD \* - р0.05 versus intact animals

\* - р0.05 versus intact animals

**Table 21.** The effect of prophylactic administration of grape wines of "Cabernet" (C) and "Rkatsiteli" (R) grades on the plasma parameters of the stress response development in rats with the neurogenic stress (in each group n=10).

However, on day 8 of administration the antioxidant and stress-protective effects of these wines were almost similar, and on the day 10 –they practically did not differ.

On days 12 and 15 there were also no differences as to the antioxidant and stress-protective action of the wines studied, which significantly reduced activation of the free radical oxidation under the action of stress normalizing the most of the indexes investigated.

Thus, the investigated wines are characterized by the high level of the antioxidant and stress-protective activity, and in the first days of introduction "Cabernet" wine improved more effectively the antioxidant status in the blood and the liver tissue than "Rkatsiteli" wine, but by day 10 the effects of the studied wines had no substantial difference.

Probably, these results are dependent on polyphenol cumulation in the organism because it is known that the polyphenol content of "Cabernet" is 10 times more than of "Rkatsiteli".

Thus, the results suggest that "Cabernet" and "Rkatsiteli" wines have already revealed the high stress-protective, hepatoprotective and anti-atherogenic activity in the conditions of the emotional-painful stress on the 2-3 days after introduction, and practically normalized the oxidative status and the lipid metabolism under the action of stress in prophylactic administration within 10 days. This indicates that grape polyphenols possess a high total antioxidant activity. At the same time the last suggestion required further research.

In order to examine the effects of wine stocks and polyphenolic concentrates obtained from other grape grades on development of proatherogenic consequences of the emotionalpainful stress we investigated the action of substances obtained from the grapes of hybrid grades "Krasen", "Golubok" and "Podarok Magaracha" produced by the National Institute of Grape and Wine "Magarach".

In the series of experiments we used purebred male rats that during 21 day were given daily*, per os,* table wine stocks of the grades "Podarok Magaracha", "Krasen" and "Golubok" in the human equivalent dose corresponding to 300 ml of wine for a human with 70 kg of the body weight. Other groups of animals were given ethanol in the human equivalent dose corresponding to 30 ml of ethanol for a human with 70 kg of the body weight taking into account the species sensitivity coefficients, as well as the table wine stocks of the grades mentioned in doses equivalent to the polyphenol content of the given wines calculated by the polyphenol content in active doses (AD – 9 mg of polyphenols/100 g of the body weight).

The results have demonstrated that not only polyphenolic concentrates, but the table wine stocks also revealed a substantial stress-protective activity to a different extent (Tables 22-25).

In fact, "Krasen" table wine stock revealed the highest activity; the stress-protective activity was almost 2.4 times more the ethanol activity in the dose studied. This product effectively prevented the activation of free radical oxidation both in the blood (increased the level of compounds with isolated double bonds in the atherogenic ApoB-LP, decreased the content of peroxidation products – diene conjugates – almost 3 times comparing to the stressed animals, and 15% - comparing to the intact animals), and the liver tissue (prevented the antioxidant content decrease, particularly the content of -tocopherol and ascorbic acid returned practically to the intact level, and there was 40% decrease of the diene conjugates level). At the same time this table wine stock prevented hyperlipidemia and the shift of metabolism to the increased lipolysis, there was 60% decrease of the blood total lipid content comparing to the stressed animals, and 11% - comparing with the intact animals. At the same time the TAG content in the liver was equal to the intact level that also demonstrated the protective action of this table wine stock. Reduction of lipogenesis in the liver tissue under the action of this product is important, and it protects the organ from steatosis. It should be also mentioned that the given product normalized the cholesterol content in the blood plasma.


The data presented as mean±SD

478 Lipoproteins – Role in Health and Diseases

of Grape and Wine "Magarach".

However, on day 8 of administration the antioxidant and stress-protective effects of these

On days 12 and 15 there were also no differences as to the antioxidant and stress-protective action of the wines studied, which significantly reduced activation of the free radical

Thus, the investigated wines are characterized by the high level of the antioxidant and stress-protective activity, and in the first days of introduction "Cabernet" wine improved more effectively the antioxidant status in the blood and the liver tissue than "Rkatsiteli"

Probably, these results are dependent on polyphenol cumulation in the organism because it is known that the polyphenol content of "Cabernet" is 10 times more than of "Rkatsiteli".

Thus, the results suggest that "Cabernet" and "Rkatsiteli" wines have already revealed the high stress-protective, hepatoprotective and anti-atherogenic activity in the conditions of the emotional-painful stress on the 2-3 days after introduction, and practically normalized the oxidative status and the lipid metabolism under the action of stress in prophylactic administration within 10 days. This indicates that grape polyphenols possess a high total

In order to examine the effects of wine stocks and polyphenolic concentrates obtained from other grape grades on development of proatherogenic consequences of the emotionalpainful stress we investigated the action of substances obtained from the grapes of hybrid grades "Krasen", "Golubok" and "Podarok Magaracha" produced by the National Institute

In the series of experiments we used purebred male rats that during 21 day were given daily*, per os,* table wine stocks of the grades "Podarok Magaracha", "Krasen" and "Golubok" in the human equivalent dose corresponding to 300 ml of wine for a human with 70 kg of the body weight. Other groups of animals were given ethanol in the human equivalent dose corresponding to 30 ml of ethanol for a human with 70 kg of the body weight taking into account the species sensitivity coefficients, as well as the table wine stocks of the grades mentioned in doses equivalent to the polyphenol content of the given wines calculated by the polyphenol content in active doses (AD – 9 mg of polyphenols/100 g of the body weight).

The results have demonstrated that not only polyphenolic concentrates, but the table wine stocks also revealed a substantial stress-protective activity to a different extent (Tables 22-25). In fact, "Krasen" table wine stock revealed the highest activity; the stress-protective activity was almost 2.4 times more the ethanol activity in the dose studied. This product effectively prevented the activation of free radical oxidation both in the blood (increased the level of compounds with isolated double bonds in the atherogenic ApoB-LP, decreased the content of peroxidation products – diene conjugates – almost 3 times comparing to the stressed animals, and 15% - comparing to the intact animals), and the liver tissue (prevented the antioxidant content decrease, particularly the content of -tocopherol and ascorbic acid returned practically to the intact level, and there was 40% decrease of the diene conjugates level). At the same time this table wine stock prevented hyperlipidemia and the shift of

oxidation under the action of stress normalizing the most of the indexes investigated.

wine, but by day 10 the effects of the studied wines had no substantial difference.

antioxidant activity. At the same time the last suggestion required further research.

wines were almost similar, and on the day 10 –they practically did not differ.

\* - р0.05 versus intact animals

\*\* - р0.05 versus stressed animals

**Table 22.** The effect of grape polyphenol concentrates and grape wines on the liver lipid metabolism in rats with the neurogenic stress (in the crude tissue, in each group n=10).

It is also necessary to point out that the control intake of the investigated substances (Tables 22-25) did not reveal negative effects on the organisms of the experimental animals. Moreover, in addition to the antioxidant activity these substances revealed a significant hypocholesterolemic and anti-atherogenic action, which was more pronounced when using "Krasen" grade wine stock and the concentrate.


The data presented as mean±SD

\* - р0.05 versus intact animals

\*\* - р0.05 versus stressed animals

**Table 23.** The effect of grape polyphenol concentrates and grape wines on the oxidant/antioxidant status in the liver tissue in rats with the neurogenic stress (in the crude tissue, in each group n=10).

Antioxidant Complexes and Lipoprotein Metabolism –

Experience of Grape Extracts Application Under Metabolic Syndrome and Neurogenic Stress 481


The data presented as mean±SD

480 Lipoproteins – Role in Health and Diseases

Group

Str.+Con. Podarok Magaracha (AD)

> Str.+Con. Krasen (АD)

Srt.+Wine Podarok Magaracha

Srt.+Wine Krasen

Str.+Con. Podarok Magaracha (DW)

Str.+Con. Krasen (DW)

Wine Podarok Magaracha

> Wine Krasen

Con. Podarok Magaracha (AD)

> Con. Krasen (AD)

> > Ethanol 5.31

The data presented as mean±SD \* - р0.05 versus intact animals \*\* - р0.05 versus stressed animals

"Krasen" grade wine stock and the concentrate.

GSH, mkmol/g

3.34 0.02\*,\*\*

3.8 0.02\*,\*\*

3.48 0.02\*,\*\*

3.57 0.08\*,\*\*

2.00 0.07\*,\*\*

> 3.27 0.06\*

4.68 0.10\*,\*\*

> 4.56 0.24

> 4.46 0.13

4.82 0.14\*

0.35\*

It is also necessary to point out that the control intake of the investigated substances (Tables 22-25) did not reveal negative effects on the organisms of the experimental animals. Moreover, in addition to the antioxidant activity these substances revealed a significant hypocholesterolemic and anti-atherogenic action, which was more pronounced when using

> -Тocopherol, nmol/g

> > 24.01 0.47\*,\*\*

> > 26.71 0.46\*,\*\*

> > 26.71 0.34\*,\*\*

28.46 0.75\*\*

21.72 0.51\*,\*\*

21.12 0.39\*,\*\*

> 33.59 0.60

35.48 0.78\*

35.29 0.45\*

27.84 0.39

24.16 1.40\*

**Table 23.** The effect of grape polyphenol concentrates and grape wines on the oxidant/antioxidant status in the liver tissue in rats with the neurogenic stress (in the crude tissue, in each group n=10).

Parameter

Diene conjugates, nmol/g

> 14.96 0.22\*,\*\*

> 15.03 0.11\*,\*\*

13.81 0.18#,\*\*

13.36 0.40\*\*

16.05

16.72 0.16\*,##

> 10.42 0.53\*

> 9.27 0.24\*

10.87 0.41

10.26 0.06\*

12.62 0.60

0.11\*,## 0.47

ТBA-active products, nmol/ mg protein

> 0.49 0,01

> 0,45 0.02

0.42 0.03\*\*

0.21 0.01\*,\*\*

0.01

0.45 0.02

0.15 0.01\*

0.13 0.01\*

0.20 0.01\*

0.15 0.01\*

0.46 0.02

Ascorbic acid, mkmol/g

> 1.21 0.01\*,\*\*

> 1.28 0.01\*,\*\*

> 1.33 0.02\*,\*\*

> 1.41 0.03\*,\*\*

> 1.02 0.03\*,\*\*

> 1.04 0.03\*,\*\*

> > 2.16 0.04\*

1.57 0.03

1.95 0.03\*

2.00 0.04\*

2.06 0.03\*

\* - р0.05 versus intact animals

\*\* - р0.05 versus stressed animals

**Table 24.** The effect of grape polyphenol concentrates and grape wines on the plasma lipid metabolism parameters and corticosterone level in rats with the neurogenic stress (in each group n=10).


The data presented as mean±SD

\* - р0.05 versus intact animals

\*\* - р0.05 versus stressed animals

**Table 25.** The effect of grape polyphenol concentrates and grape wines on the plasma oxidant/antioxidant status in rats with the emotional-painful stress (in each group n=10).
