EFFECTS OF DIET COMPOSITION AND SUPPLEMENTATION WITH SELECTED VITAMINS B ON AMOUNT AND DISTRIBUTION OF FAT TISSUE AND BLOOD CONCENTRATION OF LIPID COMPONENTS IN RAT

Mariola Friedrich¹, Joanna Sadowska^2
1 Department of Human Nutrition Physiology, Agricultural University of Szczecin, Poland
² 2 Division of Human Nutrition Physiology, Faculty of Food Science and Fisheries, West Pomeranian University of Technology, Szczecin, Poland

ABSTRACT

The experiment described in the paper focused on effects of complementary and excess diet supplementation on fatty tissue accumulation and blood triacylglycerol, total cholesterol and HDL-cholesterol levels in rats. Untreated wheat, corn grains, and barley grits in the original diet were isocalorically substituted with wheat flour, sucrose, and B vitamins.The change of the composition of the diet and its supplementation, especially excess, were found to significantly lowered diet consumption but increased accumulation of perivisceral (per 100 g food consumed) and intramuscular fat and significantly increased triacylglycerol, cholesterol and its HDL-fraction levels in the blood.

Key words: rat, supplementation, vitamins, lipids, fatty tissue.

INTRODUCTION

Circulatory disorders belong to major mortality causes among humans, both in Poland and worldwide. The risk factors include lipid metabolism disorders, their occurrence being dependent, to a large extent, on dietary factors. As is already known, it is not only the excessive amount of fat, but also the excess of simple carbohydrates or starch in refined, processed products, that enhance increased triacylglycerol synthesis. The author's own research on the effects of isocaloric substitution of dietary carbohydrates from whole grains with carbohydrates from white flour and sucrose showed a significantly higher body weight increase, relative to the unit feed consumed, to occur. Such substitution was also found to affect the amount and distribution of fat tissue, including the perivisceral fat, in the body [5]. The changes observed are thought to be caused by, i.a., deficiency of vitamins and minerals involved in lipid metabolism and in the closely related carbohydrate metabolism [12].

The increasing public awareness of dietary insufficiencies, efficient advertising as well as accessibility and convenience of 'taking care of one's health' result in more and more people using vitamin-containing formulae as supplements of daily diet [13]. Therefore it seemed interesting to investigate, on an animal model, effects of supplementation of a processed and refined diet with vitamins B involved in carbohydrate-lipid metabolism on the amount and distribution of fat tissue and concentration of lipid components in the blood.

MATERIAL AND METHODS

With the consent of the Local Ethics Commission, the experiment was carried out on 176 Wistar breed rats (males and females) aged 8 months; the females used had already produced their last brood of litter. The animals were randomly assigned to four groups 44 individuals (22 males and 22 females). Rats were fed ad libitum a pelleted feeds mixes manufactured by GRSP Miłosław. Group I received the basic mix, while Groups II, III, and IV were fed modified mixes. The modification involved substitution of the basic mix's whole wheat grains with wheat flour (type 500); 50% of the corn and 30% of the barley grits in the original mix were substituted with sucrose. The composition of feeds used in the experiment is given in Table 1.

To determine the actual chemical composition of the feeds, they were analysed for the content of crude protein [20], fat [24], dry matter [21] and ash [22]. The carbohydrate contents were calculated from the differences between dry matter contents and the sum of the remaining components. The content of dietary fibre was determined as well, in an ANKOM 220 apparatus [23] (Table 2). The gross and metabolic energy contents were calculated by means of the commonly used energy equivalents.

Group I and II drank tap water left to stand, the drinking vessels being refilled as the water was used. Group III rats (complementary supplementation) were offered, at the time of increased activity, 50 ml of aqueous solution of vitamins B_1, B₂, B₆ and nicotinic acid per unit feed weight consumed. The amounts of vitamins supplemented, i.e., 1.083 mg thiamine, 0.361 mg riboflavin, 0.898 mg pyridoxine, and 9.58 mg nicotinic acid amide per 1 kg feed were calculated from differences of their contents in the basic diet and in the modified one from which they were partly removed by component substitution. Group IV animals (excess supplementation) received, at the time of increased activity, 50 ml of aqueous solution of vitamins obtained from the over-the-counter preparation Vitaminum B-compositum (Pliva Kraków). Vitamins were given in amount: 29.4 mg of thiamine, 43.4 mg of riboflavine, 44.8 mg of pyridoxine, 364.0 mg of nicotinamide per 1 kg of fodder. The amounts of vitamins consumed by animals exceeded several times the differences between their contents in the basic diet and the modified one: 14 x for thiamine, 10 x for riboflavin, 9 x for pyridoxine and 17 x for nicotinic acid amide. Having drunk the vitamin solution, the rats drank tap water. When calculating the vitamins doses to be administered in aqueous solutions the animals's demand for vitamins has not been considered. It was take for granted that the vitamin dose in the basic diet was fully satisfactory.

The experiment took 8 weeks during which the amount of feed consumed was controlled, and the rats were weighing once a week. On termination of the experiment, the rats were anaesthetized with an anaesthetic Ketanest, their cardiac blood was collected and the amount of pericardial, peri-intestinal, and intramuscular fat was determined.

Pericardial and peri-intestinal fat was dissected out immediately after sacrificing the rats and weighed to 0.001 g.

The blood serum was used for the following assays: (i) triacylglycerols, determined with the enzymatic colorimetric technique of McGowan et al. [14], (ii) total cholesterol, determined with the enzymatic colorimetric technique of Stein [31], (iii) HDL-cholesterol, determined by elimination, a technique involving a HDL-dissolving detergent which blocks enzymatic activity of esterase and cholesterol oxidase towards VLDL- and LDL-cholesterols [15].

Intramuscular fat was obtained from the shoulder and thigh muscles (m. latissimus dorsi, m. quadriceps femoris, m. biceps femoris, m. semimembranosus, m. adductor femoris). The samples were used to determine per cent contribution of crude fat; the assays were performed with the Soxhlet technique in a Soxtec HT6 apparatus (Foss Tecator).

The data obtained were subjected to statistical treatment, using the computer software Statistica®; Duncan test and the 2-way analysis of variance (diet x sex) for independent variables were applied [30], after the confirmation of the normal distribution, at significance level α=0.05 and α= 0.01.

RESULTS

Analysis of the diets applied on rat body weight gain showed the diet involving replacement of whole cereal grains with flour and sucrose facilitated body weight increase per 100 g feed consumed, but did not affect deposition of perivisceral fat. A particular influence was exerted here by supplementation, especially the excess one, which resulted also in reduction of the feed consumed by the experimental rats (Table 3).

Table 3. Diet type and supplementation effects on body weight increments and amounts of perivisceral and intramuscular fat tissue in rats ( ± SD, n = 176)

The males exposed to excess supplementation showed the lowest body weight gain relative to 100 g feed consumed. This effect was, however, accompanied by a significantly higher – compared with the three remaining groups – accumulation of pericardial fat, and by significantly higher – compared with the non-supplemented rats – accumulation of peri-intestinal and intramuscular fat.

Analysis of vitamin supplementation effects on the female body weight increase showed the excess-supplemented females to achieve significantly higher weight gain (relative to 100 g feed consumed), compared to the females kept on the standard diet and on the diet modified and supplemented in a complementary manner. Higher body weight gain in the excess-supplementation group were accompanied by a significantly higher accumulation of perivisceral fat and by an increased content of fat in the muscle tissue (Table 3).

The effect of sex on the changes was found to be significant; significant was also the diet-sex interaction with respect to all the parameters tested (Table 3).
A change in diet composition and supplementation was found to significantly affect blood lipid components of the rats examined.

Analysis of diet composition effects on the male rat blood serum triacylglycerol concentration showed a significant increase in the concentration in the rats fed the modified diet, the effect being intensified by both supplementation types. The females showed a significant increase in triacylglycerol concentration only when excess supplementation was applied (Table 4).

The changed diet composition was found to significantly increase the blood serum total and HDL-cholesterol concentrations both in the males and the females, the effect being intensified by excess supplementation (Table 4).

Table 4. Effect of diet type and supplementation on blood concentration of chosen lipid components in rat ( ± SD, n = 176)

The excess supplementation applied was observed to reduce the HDL-cholesterol/total cholesterol ratio in the males, an increased ratio being recorded in the females; the triacylglycerol/HDL-cholesterol ratio was changed as well.

The effects of diet and sex on the type and magnitude of changes in the components studied were found to be significant; in addition, the diet-sex interaction proved significant with respect to triacylglycerol concentrations (Table 4).

DISCUSSION

Analysis of the diet supplementation applied on accumulation and distribution of fat showed the perivisceral fat deposition to have been particularly enhanced. The increased amount of fat tissue, observed in the experimental females and males, could have been related to the excess of thiamine offered. By participating in glucose transformations in the pentosephosphate pathway, thiamine contributes to the increase in NADPH used in reducing syntheses, including syntheses of fatty acids and steroids. The changes observed could have been intensified by excessive intake of pantothenic acid and biotin, responsible for the hepatic glucose increase (not compensated by glucose utilisation in tissues), which leads to glucose being incorporated into fat tissue. Accumulation of perivisceral fat could have been also enhanced by the consumption, on the part of the animals concerned, of lower amounts of feed, which reduced the uptake of not only the basic nutrients, but also those nutrients that serve a regulating function. Iron and calcium deficiencies were demonstrated to belong to the factors which stimulate deposition of fat tissue [35]. Dietary calcium deficiency trigger the release of calcitriol which accelerates lipogenesis and triacylglycerol accumulation in the muscle tissue and in perivisceral fat. Intensified accumulation of fat in the muscle tissue, coupled with dietary calcium deficiency, observed in the experiment particularly in the females consuming the lowest amounts of feed, confirms results reported by Parikh and Yanowski [18].

Adverse effects of changed composition of the diet and of supplementation, both complementary and excess, were observed also as changed concentrations of the lipid components in the rat blood.

The significantly increased triacylglycerol concentration in the male blood serum could have resulted from the presence of sucrose in the modified diet; sucrose is a source of readily available glucose and fructose, the sugars enhancing triacylglycerol synthesis. In their comparison of triacylglycerol concentrations in women and men in relation to dietary carbohydrates, Reiser et al. [25] observed higher triacylglycerol concentrations in the subjects consuming sucrose, the triacylglycerol concentrations being then much higher in men than in women. The increase in the blood serum triacylglycerol concentration is mostly due to fructose which stimulates hepatic metabolic pathways leading to intensified synthesis of fatty acids, their esterification, VLDL release, and reduction in the rate of triacylglycerol removal from the blood. In the excess-supplemented rats, fructose – a readily available substrate, and the enzymes the vitamins supplied served as co-enzymes for - led to intensification of triacylglycerol synthesis. All those processes could have been additionally stimulated by insulin secreted to blood as a consequence of the appearance of glucose, coexisting with fructose in the blood. However, the effect, although observed in rats, would not have been very obvious in healthy humans because much of the fructose formed by disintegration of sucrose is, in humans, transformed into glucose in the intestinal mucosa before it gets to the capillary circulation.

On the other hand, the increase in triacylglycerol concentration in the blood serum of the supplemented rats could have resulted from intensified synthesis of fatty acids and their accumulation in peri-intestinal fatty tissue. In their study on healthy men of various age, Walton et al. [34], who strove to determine a relationship between blood lipid components and fatty tissue distribution, showed the blood serum triacylglycerol concentrations to be positively correlated with android obesity. Accumulation of fatty tissue in the male abdominal cavity was found to be more important than the absolute body fat content for bringing about unfavourable changes in blood concentrations of lipid components. A similar relationship was reported by Haarbo et al. [10] from women.

Lipid metabolism disorders in the excess-supplemented rats could have been caused also by an increased concentration of vitamin B₆ which enhances magnesium absorption. Generally, magnesium present in tissues affects concentrations of lipids and lipoproteins in the body because, as a cofactor of numerous enzymes, magnesium plays an important part in their transformations. However, in their study on haemodialysed patients Robles et al. [26] observed patients with increased blood serum magnesium concentration to have elevated concentrations of triacylglycerols, LDL, and VLDL. Similarly, Orowicz et al. [16] demonstrated blood serum magnesium concentrations to be positively correlated with triacylglycerol concentrations, the correlations being stronger in men than in women. In the experiment described in this paper, effects of diet composition and supplementation on blood triacylglycerol concentrations were, too, more distinct in the males than in the females. Sheorain et al. [29] are of the opinion that the effect is related to the rate of triacylglycerol removal from the blood serum, the rate being slower in males than in females.

Statistical tests revealed a significant diet x sex interaction for the blood serum triacylglycerol concentrations. As opposed to the males, the females fed the modified diet did not show any increase in triacylglycerol concentration, compared to the females fed the standard feed. This can be related with a protective effect of estrogens on lipid metabolism [28].

In addition, diet composition and the method of supplementation was found to affect the blood serum total and HDL-cholesterol concentrations in the rats examined.

Replacement of whole cereal grains by white flour and sucrose resulted in a significant increase in the blood serum cholesterol content of the rats fed the modified feed, compared to those offered the standard diet. Pfeuffer and Barth [19] pointed out that cholesterolaemic effects of such a diet could have been caused by a lower amount of dietary fibre and by sucrose present in the modified feed. The presence of fibre in the diet has already been proved, beyond all doubt, to lower cholesterol concentration. A fibre-rich diet reduces fat absorption, increases removal of cholesterol and its derivatives, and inhibits cholesterol synthesis in the liver.

The significant increase in the total cholesterol in the blood serum of the excess-supplemented rats could have resulted both from intensified cholesterol synthesis and from inhibited removal of cholesterol from the circulatory system by transformation of cholesterol into derivatives, including steroid hormones. A reduced cholesterol transformation may be a consequence of effects of vitamin B supplementation on the increase in oleic and arachidonic acids in perivisceral fatty tissue lipids of rats [7]. In their study of isolated rat cells, Sarel and Widmaier [27] found a high oleic acid level to block corticosteroid synthesis and to indirectly result in blood serum cholesterol concentration increase by inhibition of cholesterol transformation. On the other hand, increased concentrations of arachidonic acid in the perivisceral fatty tissue lipids may result in inhibition of cholesterol transformation into aldosteron. This was reported from calves and rats by Elliott and Goodfriend [2] and Goodfriend et al. [8]. The mechanism of the process has not been elucidated yet, but receptors of all the steroid hormones are now known to bind also fatty acids, which results in allosteric changes in the receptor and alters its function [32]. A change in the perivisceral fatty tissue fatty acids, resulting in altered cholesterol metabolism, may thus indirectly affect glycocorticosteroid biosynthesis and activity of the hypothalamus-hypophysis-adrenal gland axis.

The increase in the total cholesterol concentration could have been triggered by stimulation of endogenous cholesterol synthesis, induced by damages in the cell membrane structure. The damages occur, i.a., during free-radical reactions, as observed in rats by Friedrich et al. [6] as a consequence of both a change in diet composition and diet supplementation with vitamins B. Unfavourable changes could have also resulted from a reduced amount of natural antioxidants in the modified diet as well as from the presence of fructose and pro-oxidative properties of niacin and pyridoxine supplied in excess. As demonstrated by Fields et al. [3], the co-existence of fructose and pro-oxidants in a diet always results in hypertriglyceridaemia and hypercholesterolaemia. Free-radical reactions could have been enhanced also by the increased accumulation of fat, including the perivisceral fat, in the excess-supplemented rats [11].

The HDL-cholesterol concentration was affected by the diet and supplementation as well. The concentration in question was observed to increase significantly in the rats kept on the modified diet; the supplementation – particularly the excess one – intensified the effect. The increase in HDL-cholesterol concentration may be ascribed to niacin which inhibits high-density lipoprotein catabolism [17].

When interpreting effects of dietary factors in the context of the threat of sclerosis, it is more useful to compare the ratio of blood serum HDL-cholesterol concentration to the total cholesterol content [4] or the triacylglycerols/HDL-cholesterol ratio, the latter being strongly correlated with the risk of coronary artery disease [1]. That was also relevant with respect to laboratory rats, although their blood lipid profile differs from that of humans in a distinct domination of high-density lipoproteins, due to a low activity of the protein vector of cholesterol esters [9]. When, however, the HDL-cholesterol/total cholesterol ratio was considered, the percentage of HDL-cholesterol in the males receiving excess supplementation was reduced, despite an overall increase in the high-density lipoproteins-bound cholesterol; an increase in the HDL-cholesterol percentage was observed in the females. The reduction of the ratio in the males is indicative of a disproportionally higher increase in their total cholesterol concentration relative to the increase in the HDL-cholesterol content. Walton et al. [34], who studied healthy men of different age, reported a negative correlation between the HDL-cholesterol concentration (and the resultant increase in the total cholesterol/HDL-cholesterol) and accumulation of fat tissue in the abdominal cavity. The triacylglycerol/HDL-cholesterol ratio was observed to change for the worse in the excess-supplemented males as well.

The sex effect on cholesterol metabolism could have resulted from a protective role of estrogens on lipid metabolism: estrogens reduce the blood serum LDL- and total cholesterol concentrations and increase the HDL-cholesterol concentration [33].

CONCLUSIONS

Analysis of the results allowed to conclude that replacement of whole cereal grains by white flour and sucrose, coupled with supplementation with selected vitamins of the B group, significantly modified lipid metabolism in rats. The modification was manifested as:

• accumulation of perivisceral fat and increased fat content in the muscle tissue of the supplemented rats, particularly the females; and
• increased concentrations of triacylglycerols, total and HDL-cholesterol in blood serum of the animals fed the modified diet, the effects being intensified by the supplementation applied, particularly in the males.

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Accepted for print: 3.09.2008

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