High conjugated linoleic acid enriched ghee (clarified butter) increases the antioxidant and antiatherogenic potency in female Wistar rats

Hypercholesterolemia, obesity and oxidation stress are the major risk factors that, alone or in combination, accelerate the development of cardiovascular diseases and the progression of atherosclerosis [1]. Due to insoluble nature of cholesterol in blood, it is transported to and from the cells by carriers known as lipoproteins- Low-density lipoprotein (LDL) and High-density lipoprotein (HDL). People with high triglyceride level along with high LDL level and a low HDL level are at health risk [2]. Antioxidants are the molecules that inhibit the oxidation of other molecules and these include glutathione, vitamin C, vitamin A, and vitamin E as well as enzymes such as catalase, superoxide dismutase and various peroxidases [3]. Water-soluble antioxidants react with oxidants (free radicals) in the cell cytosol and the blood plasma, while lipid-soluble antioxidants protect the cell membranes from lipid peroxidation.

Conjugated linoleic acid (CLA) is an important food component that consists of a mixture of positional and geometric isomers of linoleic acid (cis − 9, cis − 12, C_18:2) with two conjugated double bonds at various carbon positions in the fatty acid chain [4]. CLA is formed as an intermediate during the biohydrogenation of linoleic acid by linoleic acid isomerase from the rumen bacteria Butyrivibrio fibrisolvens[5] or from the endogenous conversion of t-11, C_18:1 (vaccenic Acid), another intermediate of linoleic or linolenic acid biohydrogenation, by Δ⁹-desaturase in the tissues [6]. Milk fat is the richest natural dietary source of CLA which contains an average 4.5 mg CLA/g of fat [7]. CLA is considered as an important biologically-active compound of food due to its proven anticarcinogenic, antiallergic and anti-inflammatory properties [8, 9].

The relationship between food and health has long been known to exist, and today the fundamental concept of food is changing from one involving the maintenance of life to one maintaining and promoting better health and quality of life by preventing chronic diseases. As mentioned earlier, hypercholesterolemia, obesity and oxidative stress are the major risk factors for developing atherosclerosis and cardiovascular diseases in humans. Although numerous synthetic drugs are available for decreasing cholesterol levels [10], still there are a lot of side effects associated with these drugs. This anomaly has renewed research interests in developing new strategies to use natural components for maintaining cholesterol levels with least side effects. Hence, taking these factors into consideration, the aim of the present work was to study the effect of CLA enriched ghee (clarified butter) on the antioxidant enzyme activities of blood and liver of CLA fed Wistar rats. In addition to this, antiatherogenic activity in aorta and liver of Wistar rats was also studied for maintaining health.

The objective of this study was to assess the effect of CLA containing food product (ghee) on antioxidative enzyme system and to study the antiatherogenic property in the blood and liver of rats. The results indicated that feeding of low and high CLA ghee diets during pubescent period (from 21^st day to 55^th day) increased 20 & 33% CAT and 13 & 28% SOD activity, respectively, in blood as compared to the group fed on soybean oil diet. Similarly, an increased liver CAT and SOD activities were observed in high CLA group as compared to soybean oil fed group. Hence, higher CAT & SOD activities in group fed with high CLA ghee diet may offer higher protection to the cells against oxidants. Similar results were reported by Santos Zago et al., [11], wherein they showed that administration of CLA to the diet of rats increased the catalase and lipid peroxidases, which finally lead to an improvement in the antioxidative process. Higher CAT and SOD activities in response to CLA diet given to rats have also been reported by Ip et al. [12] and Bhatia [13]. Thus, in our work, ghee with high CLA has enhanced the activity of CAT and SOD enzymes by scavenging the free radicals which ultimately improves the antioxidative enzyme systems. In pubescent development of rats, the level of nitric oxide was observed to be the same in all the treatment groups. Not much significant variations were observed in the treatment groups; which clearly shows that nitric oxide is independent of CLA containing diet. Haemoglobin, too, is not much affected by the CLA content in the diet. Not much significant effect on haemoglobin was observed by Steck et al. [14] in case of rats administered with CLA content.

In the present study, GST activity was the highest in high CLA ghee followed by low CLA ghee fed groups rather than in soybean oil fed group. Higher GST activity in response to high CLA are believed to modify the antioxidant system in rats [15, 16].

Feeding of low and high CLA ghee from weaning to sexual maturity of rats lowered 27 and 40% lipid peroxidation than the soybean oil fed rats in present study. Lipid peroxidation is considered as the main molecular mechanisms involved in the oxidative damage to cell structures and in the toxicity process that lead to cell death [17]. Oils have been reported to induce a significant increase in the antioxidant enzyme activities and a decrease in the conjugated dienes (CD) and thiobarbituric acid-reactive substances levels in the liver [18]. Thus high CLA fed diet lowers the lipid peroxidation process and inturn protects the cells from its harmful effects. Recent study by Varady et al. [19] has proved that oxidized fat increases the nuclear concentration of NF-κB and Nrf2 and transcript levels of oxidative stress-responsive genes coding for aldo–keto reductase 1B8, vanin-1, glutathione peroxidase 1, and superoxide dismutase-1. Hence, CLA plays a definite role in taking care of oxidative stress in rats and removing the free radicals from the biological system.

Several animal studies have established that CLA has profound effects on lipoprotein metabolism and it prevents diet-influenced atherosclerosis. Hence the experiment was conducted to study the antiatherogeneic potency of CLA in adult female (9 weeks old) rats by feeding CLA for 120 days. Not much significant variations in the average body weight of rats fed with different dietary treatments were observed. This proves that CLA has anti-obesity property, which may be attributed to the modulation in lipid metabolism leading to the reduction of lipid uptake by adipocytes and manifested as reduction in body fat gain [2]. Yu et al. [20] reported that two weeks feeding of trans-10, cis-12 CLA reduced abdominal adipose tissue weight than linoleic acid fed to rats. These effects were attributable to suppressed fatty acid synthesis and enhanced beta-oxidation of fatty acid in the liver on trans-10, cis- 12 CLA diet.

In our study, low and high CLA feeding reduced total cholesterol by 52.17 and 26.06%, triglycerides by 23.00 and 11.00%, however, HDL increased by 17.82 and 33.26% than soybean oil feeding in rats. The supplementation of diet containing CLA has shown a reduction in the levels of serum total cholesterol and total triglyceride and increased high-density lipoprotein cholesterol level as compared to control diet groups without CLA [21, 22]. In another study by Bauchart et al. [23] butters varying in trans 18:1 and cis-9,trans- 11 Conjugated Linoleic Acid has modified Plasma Lipoproteins in the Hypercholesterolemic Rabbit. Present results clearly indicated that CLA feeding, irrespective of its level in diet, narrowed the atherogenic index (0.472 to 0.244) in rats than those fed on soybean oil (0.410 to 0.971) by decreasing LDL and increasing HDL.

Our results also indicated that the presence of CLA in the diet of rats inhibited deposition of cholesterol and triglycerides by two and three folds, respectively, in liver as well as in aorta and the response of CLA was depending upon its level in the diet. Fatty acid composition and presence of polar lipids in the diets have significant effects on lipid partitioning, gene expression, and potentially the development of liver pathology in mice [24]. In a recent study by Tan et al. [25], they have proved that CLA addition significantly increases the Carnitine palmitoyltransferase I enzyme levels (that catalyzes an important regulatory step in lipid metabolism) which ultimately results in the reduction of lipid contents both in liver and muscle tissues.

As discussed earlier, dietary CLA tends to modulate lipid metabolism in rats. In the present study, soybean oil feeding resulted in more fat deposition in hepatocytes compared to the CLA feeding (Plate 4.1B). Recent work by Kostogrys and Pisulewski [26] have also discussed about the alterations in the histopathology of liver tissues and concluded that CLA in high-fructose diet, decreases serum LDL + VLDL and trigylcerides and plasma malondialdehyde concentrations as well as liver weight and liver cholesterol, thus opposing the effects of high-fructose diet and showing a potential antiatherogenic effect.

The study shows that high CLA enriched ghee increases the antioxidant enzyme activities such as CAT, SOD and GST activities; which concludes its antioxidant nature. Feeding of high CLA enriched ghee also lead to a decrease in cholesterol, triglyceride and high density lipoproteins and inturn, decreased the low density lipoproteins, which finally lead to the reduction in atherogenic index, which proves its antiatherogenic property. The present study indicates that high CLA enriched ghee (clarified butter) has antioxidant and antiatherogenic activities suggesting that ghee can be as an important food component for decreasing the risk of cardiovascular diseases.

Protocols were conducted in accordance with standard ethical guidelines, it was approved by the IAEC (Institutional Animal Ethics Committee).