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Abstract

The present study was undertaken to investigate the effects of the combination of niacin and chromium(III)-chloride on heart glutathione (GSH), lipid peroxidation (LPO) levels, serum paraoxonase (PON), gamma-glutamyl transferase (GGT) activities and protein carbonyl contents (PCC) of hyperlipidemic rats. In this study, female Swiss albino rats were used. They were divided into four groups. The animals of the first group (group I) were fed with pellet chow. The rats (group II) were fed with a lipogenic diet consisting of 2% cholesterol, 0.5% cholic acid and 20% sunflower oil added to the pellet chow, and given 3% alcoholic water for 60 days. The rats (group III) were fed with the same lipogenic diet and treated by gavage technique with CrCl₃ 6H₂O to a dose of 250 µg/kg and 100 mg/kg niacin for 45 days, 15 days after experimental animals were done hyperlipidemic. Group IV was fed with pellet chow and treated with 250 µg/kg CrCl₃ 6H₂O and 100 mg/kg niacin for 45 days. On the 60th day, the heart tissue and blood samples were taken from animals. As a result, heart LPO, serum GGT activity and serum PCC were increased; serum PON activity and heart GSH levels were decreased in hyperlipidemic rats. Treatment with combined niacin and chromium reversed these effects. In conclusion, the combined treatment with niacin and chromium might induce a protective effect on heart tissue.

Keywords

niacin chromium hyperlipidemia serum heart

Introduction

Hyperlipidemia is one of the major risk factors for cardiovascular disease.¹ In the world, coronary heart disease is one of the most important causes of death as in Turkey. Therefore, to prevent the development of this disease is an important issue in terms of public health. Approximately 12 million people reportedly die of cardiovascular disease and cerebral apoplexy each year worldwide. For these reasons, finding methods for early prevention and control of hyperlipidemia is crucial. Most current hypolipidemic drugs are expensive and have potential side effects, so research is increasingly focusing on natural drug that reduce blood lipid levels.²

Niacin (nicotinic acid) is a water-soluble vitamin of the B group.³ It is an essential nutrient required for the proper metabolism of carbohydrates, lipids and proteins. It also supports proper blood circulation and healthy skin and aids in the treatment of lipid disorders. Niacin regulates circulating lipoproteins. It might reduce long-term mortality in patients with cardiovascular disease.^4–6

Chromium is an essential dietary nutrient and plays a vital role in carbohydrate metabolism, insulin metabolism and balance, enzyme activity related to the metabolism of glucose, transport of proteins and the synthesis of fatty acids and cholesterol.^7,8 It also helps to regulate blood pressure and cholesterol and is essential for arterial health. Insufficient dietary intake of chromium leads to signs and symptoms that are similar to the ones observed for diabetes and cardiovascular diseases.

There are numerous studies related to only niacin^9–11 and only chromium treatments.^12,13 But, a few studies have been reported on the effects of combined treatment with niacin or niacin-bound chromium and other substances.^14,15 No reported studies related to the combined treatment with niacin and chromium on heart tissue could be found.

The aim of this study was to investigate the biochemical effects of combination of niacin and chromium(III)-chloride on the extent of lipid peroxidation (LPO) and glutathione (GSH) levels in heart tissues and serum paraoxonase (PON) and gamma-glutamyl transferase (GGT) activities and protein carbonyl contents (PCC) of hyperlipidemic rats.

Materials and methods

One-year-old female Swiss albino rats were used. The rats were housed in metal cages maintained at room temperature and were fed standard animal food and tap water. The experiment was reviewed and approved by the Animal Care and Use Committee of Istanbul University. In this study, the rats were divided into four groups. The animals of the first group (group I) were fed with pellet chow. The rats (group II) were fed with a lipogenic diet consisting of 2% cholesterol, 0.5% cholic acid and 20% sunflower oil added to the pellet chow and given 3% alcoholic water for 60 days. The rats (group III) were fed with the same lipogenic diet and treated by gavage technique to rats to a dose of 250 µg/kg CrCl₃ 6H₂O and 100 mg/kg niacin for 45 days, 15 days after experimental animals were done hyperlipidemic. Group IV were fed with pellet chow and treated with 250 µg/kg CrCl₃ 6H₂O and 100 mg/kg niacin for 45 days. On the 60th day, the heart tissue and blood samples were taken from animals under ether anesthesia. Heart tissues were washed with saline and frozen until needeed. Serum PON activity was measured according to Szasz.¹⁶ GGT activity was assayed according to the method used by Furlong et al.¹⁷ PCC was determined by the method of Levine.¹⁸ Heart tissues were homogenized in cold with 0.9% saline with a glass homogenizer to make up a 10% (w/v) homogenate. The homogenates were centrifuged and the clear supernatants were used for determination of GSH and LPO levels. GSH and LPO levels were determined by the methods of Beutler¹⁹ using Ellman’s reagent and Ledwozyw et al., respectively.²⁰ The protein content in the supernatant was estimated by the Lowry method using bovine serum albumin as standard.²¹

Statistical analysis

The results were evaluated using an unpaired t-test and analysis of variance (ANOVA) using the NCSS statistically computer package.

Results

The mean serum PON, GGT activities of the four groups are given in Table 1 . A significant difference in the serum PON activities were found among the four groups (p _ANOVA = 0.007). There was a significant decrease in PON activities in hyperlipidemic group compared to control groups (^bP_t-test = 0.001). Supplementation with niacin and chromium caused a significant increase in PON activity in hyperlipidemic rats (p _t-test = 0.001).

Table 1.

Serum paraoxonase (PON), and gamma–glutamyl transferase (GGT) activities and protein carbonyl contents (PCC) for all groups

Groups	PON (U/L)^a	p _t-test	GGT (U/L)^a	p _t-test	PCC (U/L)^a	p _t-test
Control Control+niacin+Cr	112.06 ± 45.36 123.76 ± 66.44	0.687	2.99 ± 1.13 5.37 ± 0.52	0.026	19.67 ± 2.54 26.53 ± 15.94	0.502
Hyperlipidemia Hyperlipidemia+niacin+Cr	46.39 ± 13.67^b 121.95 ± 53.12	0.001	7.22 ± 2.74^c 1.98 ± 1.60	0.046	30.25 ± 3.46^d 19.50 ± 2.41	0.005
p _ANOVA	0.007		0.007		0.413

^a Mean ± SD.

^b p _t-test = 0.001 versus control group

^c p _t-test = 0.007 versus control group

^d p _t-test = 0.068 versus control group

Serum GGT activities in hyperlipidemic group were significantly increased as compared to the control group (^c p _t-test = 0.007). A decrease of the GGT activities in hyperlipidemic group treated with niacin and chromium was observed (p _t-test = 0.046).

There was no significant difference in serum PCC levels between groups (p _ANOVA = 0.413; Table 1). Serum PCC levels in hyperlipidemic group were increased as compared to control group (^d p _t-test = 0.068). Supplementation with niacin and chromium caused a significant decrease in serum PCC levels of hyperlipidemic rats (p _t-test = 0.005).

Heart GSH and LPO levels of four groups are shown in Table 2 . The GSH levels were decreased in the heart tissue of hyperlipidemic rats, compared to controls (^b p _t-test = 0.0001). In the hyperlipidemic rats treated with niacin and chromium, the heart GSH levels significantly increased when compared to the hyperlipidemic group (p _t-test = 0.001). A significant difference in heart GSH levels of four groups was observed (p _ANOVA = 0.0001) (Table 2). The LPO levels were increased in the heart tissue of hyperlipidemic rats, compared to controls (^c p _t-test = 0.001). The heart LPO levels of hyperlipidemic rats given niacin and chromium significantly decreased when compared to that of the hyperlipidemic group (p _t-test = 0.002; Table 2).

Table 2.

Heart tissue glutathione (GSH) and lipid peroxidation (LPO) levels for all groups

Groups	GSH (nmol GSH/mg protein)^a	p t-test	LPO (nmol MDA/mg protein)^a	p t-test
Control Control+niacin+Cr	14.30 ± 4.81 13.85 ± 7.06	0.872	1.19 ± 0.42 1.27 ± 0.76	0.749
Hyperlipidemia Hyperlipidemia+niacin+Cr	5.73 ± 1.72^b 12.59 ± 5.32	0.001	3.72 ± 2.15^c 1.19 ± 0.79	0.002
p _ANOVA	0.004		0.0001

^a Mean ± SD.

^b p _t-test = 0.0001 versus control group.

^c p _t-test = 0.001 versus control group.

Discussion

Hyperlipidemia may cause cardiovascular disease, renal disease and some medical problems following solid organ transplantation.^22,23 The most prominent risk factors for cardiovascular disease are related to diet and lifestyle.^24,25 Regarding dietary habits, foods consumed in industrialized countries are usually overly rich in saturated fats and cholesterol.^24,25 Hyperlipidemia induces oxidative stress through various mechanisms. Oxidative stress exerts cytotoxic effects by peroxidation product malondialdehyde (MDA), leading to changes in cell permeability, integrity and, ultimately, cell death.²⁶ Oxygen free radical damage is usually prevented by a series of nutritional supplements that include vitamins C (ascorbic acid), E (tocopherols), A (carotenoids) and B₃ (niacin) and the trace elements chromium and selenium.^27,28 There are also specific biological defense mechanisms that protect tissues against cellular damage.

Niacin has been widely used clinically to regulate abnormalities in lipid / lipoprotein metabolism and in the treatment of atherosclerotic coronary heart disease. Clinical studies have demonstrated that niacin alone or in combination can slow or reverse the progression of atherosclerosis and reduce cardiovascular event rates and total mortality in patients with hypercholesterolemia and established atherosclerotic cardiovascular disease.²⁹

Chromium is an essential element for carbohydrate and lipid metabolism in animals and humans.³⁰ Some researchers highlighted its role with regard to the metabolism of lipids and the correlations between chromium status and cardiovascular disease.³¹ Low chromium levels lead to high cholesterol and bring the risk of developing cardiovascular disorders.

PON is a serum enzyme, which prevents oxidation of low-density lipoprotein by hydrolyzing lipid peroxides.³² Lipid oxidation may play an important role in the development of micro- and macrovascular diseases. The oxidation of lipids by free radicals results in reactive products such an aldehydes, ketones and hydroxy acids. Direct interaction of these compounds with DNA has been shown to cause genetic damage and disturbance of cell signaling in human cancers, neurodegenerative diseases and atherosclerosis.³³ The PON hydrolyzing lipid peroxides inhibits the development of further oxidative possesses and thus, prevents the oxidative damage of DNA. Harangi et al. have reported that oxidative DNA damage defected by comet assay was increased in hyperlipidemic patients and was associated with the decrease in PON activity.³⁴ The activity of PON 1 can be influenced by acquired factors such as diet, life style and diseases. Serum PON 1 activity was reduced in some clinical disorders such as coronary artery disease³⁵, familial hypercholesterolemia³⁶ and hyperlipidemia.³⁷ Increased oxidative stress has been shown to reduce PON1 synthesis in animal and cell culture models.³⁷ This decrease in PON1 activity in serum was related to degree of liver and other tissue damage. In the present study, serum PON activity was significantly low in the hyperlipidemic groups. The decrease in PON activity represents increased utilization due to oxidative stress and LPO. Administration of combined niacin and chromium significantly increased serum PON activity in the hyperlipidemic group showing that this treatment had prevented hyperlipidemia-induced oxidative stress and LPO and that PON1 activity in serum has fallen to normal levels.

GGT is a key enzyme in the catabolism of GSH. It has been reported that the extracellular cleavage of GSH by GGT induces the production of ROS, suggesting that GGT plays a pro-oxidant role.³⁸ Therefore, the present results suggest that serum GGT might be one of the enzymes related to oxidative stress after hyperlipidemia. In our study, serum GGT activity was markedly elevated in hyperlipidemic animals compared to control rats. Combined niacin and chromium caused a significant decrease in the serum GGT activity in hyperlipidemic rats. These effects could be due to a protective antioxidant effect of niacin and chromium.

Protein carbonyl content is the most general indicator and by far the most commonly used marker of protein oxidation.³⁹ Thiols, including glutathione, are protective antioxidants acting as free radical scavengers; therefore, the thioldisulfide balance can be an indication of oxidative damage in tissue. Significant increase in the protein carbonyl content was observed in hyperlipidemic rats. Oxidative modifications alter the biological properties of proteins, leading to their fragmentation. Administration of niacin and chromium to hyperlipidemic rats decreased the protein carbonyl levels indicating that it is able to reduce protein oxidation.

Rodriguez-Porcel et al. demonstrated that experimental hypercholesterolemia is associated with blunted myocardial perfusion and increased vascular permeability responses in vivo to increased cardiac demand.⁴⁰ This cardiac demand is associated with increased markers of oxidative stress and depleted tissue endogenous oxygen radical scavengers and antioxidants. LPO is regarded as one of the basic mechanisms of cellular damage caused by free radicals.⁴¹

Oxidative stress is a reflection of excess intracellular concentration of oxidants, such as H₂O₂ and O₂ ^- as well as antioxidants defense molecules such as GSH. GSH, as one of the reduced thiol agents, is capable of interacting with free radicals to yield more stable elements and is known for its ability to repair membrane lipid peroxides.⁴² The level of GSH significantly decreased in cases of oxidative stress.⁴³ Low levels of GSH are associated with a number of disease conditions known to generate high amounts of ROS, such as observed in atherosclerosis, heart failure, diabetes and neurodegenerative disorders.⁴⁴ In this study, the GSH levels were decreased in heart tissue of hyperlipidemic rats, compared to controls. Depletion of GSH in the heart tissue of hyperlipidemic rats may be due to scavenging of lipid peroxide. Administration of niacin and chromium increased the content of GSH in heart of hyperlipidemic rats, showing the beneficial effect of the treatment on ROS.

Hyperlipidemia leads to increased production of oxygen free radicals,⁴⁵ which exert their cytotoxic effects by causing LPO and the resulting formation of MDA. Others studies have reported a rise in the LPO levels in animals administered with a high-cholesterol diet.⁴⁶ Heart tissue has been affected by hyperlipidemia. The LPO levels were significantly increased in heart tissues of hyperlipidemic groups. Under normal conditions, LPO is controlled by the body’s antioxidant defense mechanisms. In this study, enhanced LPO in heart tissues of hyperlipidemic rats may be attributed to excessive generation of ROS. This study shows that administration of combined niacin and chromium to hyperlipidemic rats prevents the tissue accumulation of LPO. In another study carried out in our laboratory, we have observed that skin, lung, liver and other tissues LPO was increased in hyperlipidemic rats.⁴⁷ On the other hand, treatment with niacin and chromium reversed these effects.

In conclusion, the present study revealed that the combined treatment with niacin and chromium in hyperlipidemic rats might induce a protective effect on antioxidant defense system in the heart tissue.

Footnotes

This work was supported by Scientific Research Projects Coordination Unit of Istanbul University: BYP-9667.

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Combined effects of niacin and chromium treatment on heart of hyperlipidemic rats

Abstract

Keywords

Introduction

Materials and methods

Statistical analysis

Results

Discussion

Footnotes

References