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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 58:B788-B791 (2003)
© 2003 The Gerontological Society of America

Effect of DL-{alpha}-Lipoic Acid on the Status of Lipid Peroxidation and Lipids in Aged Rats

Palaniyappan Arivazhagan1, Saraswathi R. Panneerselvam2 and Chinnakkanu Panneerselvam1

1 Department of Medical Biochemistry, Dr. ALM Post Graduate Institute of Basic Medical Sciences
2 Department of Biochemistry and Molecular Biology, University of Madras, Chennai, India.


   Abstract
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 Abstract
Methods
 Results
 Discussion
 References
 
The effect of dextro and levo (DL)-{alpha}-lipoic acid on lipid peroxidation and lipids has been evaluated in plasma, liver, and kidney of young and aged rats. The levels of thiobarbituric acid reactive substances (TBARS) and lipids were considerably higher in aged rats compared with younger controls. DL-{alpha}-lipoic acid (100 mg/kg body wt/day) was administered intraperitoneally for 7 and 14 days. Supplementation of lipoic acid in aged rats prevents the elevated levels of TBARS and lipids. From our observations, we conclude that lipoic acid is very effective in normalizing age-related alterations in lipids, and it can be implemented in the aged to minimize age-associated disorders where free radicals are the major cause.

LIPID peroxidation is characteristically a free radical chain reaction initiated by the abstraction of a hydrogen atom from a polyunsaturated fatty acid side chain. The free radical-mediated lipid peroxidation has been proposed to be critically involved in several disease states including cancer, cardiovascular diseases, and cataracts as well as the degenerative processes associated with ageing (1).

Ageing is a physio(patho)logical phenomenon associated with changes in various biological processes that disorganize homeostasis, thus leading to an ever-increasing likelihood of disease and death. Ageing involves a process of enhanced peroxidative stress resulting in oxidation of proteins, damage to DNA, and alteration of membrane lipids such as cholesterol, triglycerides, and phospholipids (2,3).

A number of enzymatic and nonenzymatic antioxidants play an important role to protect living organisms against free radical-mediated cellular damage. Our earlier investigations demonstrate that these antioxidant defense systems are found to be decreased during ageing process (4–6). One such defense system is lipoic acid, which is an essential cofactor (as lipoamide) in the oxidative decarboxylation of {alpha}-ketoglutarate. Recently, it has been suggested that, in addition to its well-recognized catalytic function, the lipoic/dihydrolipoic acid couple is involved in thiol-dependent protection against oxidative damages by interacting with other intracellular antioxidants (7,8). In addition, its action may be well extended to certain reactions in lipid biosynthesis where it replaces coenzyme A (CoA) for activating fatty acids prior to acylation (9). A decrease in the level of lipoic acid has been reported in discrete brain regions of rats during ageing (10). In this regard, the present study was undertaken to determine the effect of dextro and levo (DL)-{alpha}-lipoic acid supplementation on the status of lipid peroxidation and lipids in aged rats.


   METHODS
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 Abstract
 Methods
Results
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DL-{alpha}-lipoic acid was purchased from Sigma Chemical Company (St. Louis, MO). All other chemicals were of reagent grade. Male albino rats of Wistar strain weighing approximately 130–160 g (young) and 380–410 g (aged) were used. The animals were divided into 2 major groups. Group I consisted of normal young rats (3–4 months old) and Group II consisted of normal aged rats (above 22 months old). Each group was further subdivided into 3 groups, 1 control group (Groups Ia and IIa) and 2 experimental groups, based on the duration of lipoic acid administration for 7 days (Groups Ib and IIb) and 14 days (Groups Ic and IIc). The animals were maintained on commercial rat food that contained 5% fat, 21% protein, 55% nitrogen-free extract, and 4% fiber (wt/wt) with adequate mineral and vitamin content. Each group consisted of 6 animals and had access to food and water ad libitum. DL-{alpha}-lipoic acid (100 mg/kg body wt/day) was dissolved in 0.5% NaOH in physiological saline and administered intraperitoneally to the experimental animals for 7 and 14 days, whereas control young and aged rats received vehicle alone in a similar manner.

On completion of 7 and 14 days of lipoic acid administration, the animals were killed by cervical decapitation. Blood was collected in heparinized tubes, and plasma was separated by centrifugation. Liver and kidney tissues were excised immediately and immersed in ice-cold physiological saline. Tissue lipids were extracted by the method of Folch and colleagues (11). Lipid peroxidation was assayed by the method of Ohkawa and colleagues (12). Cholesterol was estimated by the method of Parekh and Jung (13). Triglycerides were assayed by the method of Rice (14). Estimation of phospholipids was carried out by the method of Rouser and colleagues (15). Free fatty acid was assayed by the method of Horn and Menahan (16).

Statistical Analysis
Values are expressed as mean ± SD (standard deviation) for 6 rats in each group, and significance of the differences between mean values were determined by one-way analysis of variance (ANOVA) coupled with Student-Newman-Kuel multiple comparison test. Values of p <.05 were considered to be significant.

Statistically significant differences between the young, aged, and lipoic acid-treated groups were determined by Student's t test. The levels of significance were evaluated with p values.


   RESULTS
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 Abstract
 Methods
 Results
Discussion
 References
 
Table 1 depicts the level of thiobarbituric acid reactive substances (TBARS) in plasma, liver, and kidney of young and aged rats. A significant increase in the level of TBARS (p <.001) was observed in aged rats (Group IIa) when compared with young control rats (Group Ia). The level of TBARS reverted to near normal after lipoic acid treatment in aged rats (Group IIc), compared with respective control group (Group IIa). In young rats, lipoic acid supplementation exhibited a lowered level of lipid peroxide.


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  		Table 1. Effect of DL-{alpha}-Lipoic Acid on Lipid Peroxidation in Young and Aged Rats.

 
The data presented in Table 2 show the level of cholesterol, triglycerides, phospholipids, and free fatty acids in plasma, liver, and kidney of young and aged rats. In aged rats (Group IIa), the levels of lipids were found to be higher (p <.001) compared with younger controls (Group Ia). On administration of lipoic acid to aged rats (Group IIc), the levels of lipids were lowered considerably compared with aged control rats (Group IIa). In young rats (Group Ic), the level of cholesterol decreased moderately after lipoic acid administration.


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  		Table 2. Effect of DL-{alpha}-Lipoic Acid on Plasma, Liver, and Kidney, Lipids in Young and Aged Rats.

 

   DISCUSSION
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 Results
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References
 
Lipids are essential constituents of living cells. They function as an important structural component of cell membranes, as a primary source of energy, and as a precursor for biologically potent steroid hormones, prostaglandins, leukotrienes, thromboxanes, and lipoxins. Like other biomolecules, lipids are constantly being synthesized and degraded so that their steady-state levels are maintained within certain limits. When enzymes involved in lipid metabolism are defective, lipid disorders may develop. Age-related lipid alterations have long been an area of interest for investigators seeking clues on the progressive cellular injury that occurs in aging organs (17).

Peroxidation of lipids has been suggested to be a major cause of the ageing process (1). Lipid peroxidation contributes greatly to the loss of membrane fluidity in aged animals through the effects of their hazardous radicals. In the present study, the level of lipid peroxidation was significantly higher in aged rats when compared with the young control rats. Lipids act as a vital substrate for lipid peroxidation, and enhancement of lipid profile has been reported during ageing (3). A possible reason for the observed increase in the status of lipid peroxidation in aged rats can be due to an elevation in the levels of lipids as well as increased production of free radicals during ageing. Liu and colleagues (18) reported that lipoic acid supplementation reduced the lipid peroxidation status in the brain of old rats. In addition, lipoic acid supplementation has been found to be effective in improving mitochondrial function by decreasing oxidative damage (19).

Administration of DL-{alpha}-lipoic acid to aged rats inhibits lipid peroxidation through its antioxidant action. An earlier report shows that lipoic acid is a powerful scavenger of hydroxyl radicals and can inhibit iron-dependent .OH generation (20,21). Duration-dependent protection is rendered by lipoic acid against age-related TBARS production, and this can be attributed to the two sulphydryl moieties of lipoic acid.

Cholesterol, a structural component of the membrane, is involved in maintaining the integrity of the cells and in regulating the fluidity of the cell membrane. In the present study, a marked increase in the level of cholesterol was observed in aged rats. 3-hydroxy 3-methyl glutaryl (HMG) CoA reductase, a rate-limiting enzyme in cholesterol synthesis, was shown to be inhibited by adenosine triphosphate (ATP) through an irreversible binding (22). As depletion of ATP occurs with advancing age, it might have failed to inhibit the enzyme activity and thus could have improved the cholesterol synthesis. Administration of lipoic acid to aged rats reduced the level of cholesterol in the present investigation. DL-{alpha}-lipoic acid inhibits protein oxidative modification of human low-density lipoprotein (LDL) and reduces plasma cholesterol levels by the inhibition of hepatic HMG-CoA reductase (21). Reports by Angelucci and Corandolis (23) and Krichevsky (24) also highlight the anticholesterol property of DL-{alpha}-lipoic acid. Hence it is considered to be a cardiovascular risk-lowering agent (24).

In the present study, an increase in the content of triglycerides was observed in aged rats. Impaired fatty acid oxidation diverts the free fatty acids towards increased triglyceride synthesis, lipoprotein synthesis, and fat storage (25). This may be the reason for the increase in the levels of LDL and VLDL with age (26), the consequence of which may reduce the activity of lipoprotein lipase and enhance the level of triglycerides. An age-related decrease in ascorbic acid concentration may be a contributory factor for reduction in the activity of lipoprotein lipase (27). Lipoic acid-supplemented aged rats showed a reduction in the levels of triglycerides, and this may be attributed to an increase in the activity of lipoprotein lipase (28), which in turn stimulates the clearance of triglycerides (29). DL-{alpha}-lipoic acid is known to enhance ascorbic acid formation (30). In addition, as lipoic acid can spare ascorbic acid, this might have indirectly stimulated the ascorbic acid-dependent lipoprotein lipase activity.

An age-related increase in free fatty acid content has been noticed in the present study. This may be due to its release from the membrane phospholipid pool through the activation of phospholipase A2 (PLA2) by extracellular calcium (Ca2+), which is enhanced during age-associated free radical generation (31). Another correlation can be deduced from the study of Burch (32), where the two forms of phospholipase A2, namely the phosphorylated (inactive) and dephosphorylated (active) forms, have been explained. PLA2 is regulated post-translationally by both phosphorylation and calcium. Calcium plays a role by promoting the binding of PLA2 to the membrane for access to phospholipid substrate, and this is mediated by calcium–phospholipid-binding domain (C2 domain) at the amino terminus of the enzyme (33). The carboxy-terminal part of protein kinase C (PKC) contains the protein kinase domain (C4) and an ATP-binding domain (C3) (34). The ATP level may also maintain PLA2 activity by regulating phosphorylation through protein kinases and dephosphorylation through phosphatases. A normal ATP level maintains the phosphorylated form and thus switches off phospholipase A2. Clapham (35) reported that lipid peroxidation and related Ca2+ overload inhibits the ATP production at the mitochondrial level. As a result, an age-related decline in the level of ATP leads to the dephosphorylation/unphosphorylation of the enzyme PLA2, which subsequently gets activated.

Lipoic acid administration to aged rats reduced the level of free fatty acids. Lipoic acid is known for revitalizing ATP production (36) for the phosphorylated form of phospholipase A2. Another possible reason is the sequestration of Ca2+ by lipoate (37), which indirectly reduces oxidative damage to membrane structures. In addition, McCarty (25) demonstrated that lipoic acid promotes free fatty acid oxidation and thereby reduces the concentration of free fatty acids.

Conclusion
Lipoic acid as a thiol compound may contribute its sulfhydryl groups to lower age-related lipid peroxidation and thereby protect the cell membrane from further peroxidative stress. The lipid-lowering effect of lipoic acid is important from the therapeutic point of view, as hyperlipidemia represents a consequence of ageing, the main risk of which is coronary heart disease and other arteriovascular diseases.


   Acknowledgments
 
The present study was supported by the Indian Council of Medical Research, New Delhi, India.

Address correspondence to Dr. Chinnakkanu Panneerselvam, Department of Biochemistry, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai – 600 113, India. E-mail: panneerselvam{at}eth.net


   Footnotes
 
Decision Editor: James R. Smith, PhD

Received July 18, 2002

Accepted May 13, 2003


   References
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 Methods
 Results
 Discussion
 References
 

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