In vitro toxicity of weight-loss supplements conjugated linoleic acid Levo-carnitine and hydroxycitric acid

Abstract

South Africa is experiencing a speedy epidemiologic transition with an alarming increase in obesity and associated disease. The appeal of over-the-counter dietary supplements as a “magic bullet” for weight loss entices many patients who desire to lose weight. The aim of this study was to provide evidence regarding the effect of three common weight loss dietary supplements or ingredients, and these are conjugated linoleic acid, L-carnitine and hydroxycitric acid at the daily recommended dosage. The antioxidant activity (chemical and cellular), toxicity (reactive oxygen species induction, cellular viability, erythrocyte haemolysis), effects on lipid accumulation (differentiated and differentiating adipocytes) and blood coagulation was determined using ephedrine as a weight loss control. The chemical and cellular oxidative/antioxidant effects of ephedrine, conjugated linoleic acid, L-carnitine and hydroxycitric acid were determined at low (0,75, 1, 2 and 4 _g/mL) and high (25, 50 and 250 _g/mL) concentrations with the oxygen radical absorption capacity assay. The cellular antioxidant effects of ephedrine, conjugated linoleic acid, L-carnitine and hydroxycitric acid were determined at low (7,5 – 42,5 _g/mL) and high (250 – 2500 _g/mL) concentrations with cellular 2’,7’-dichlorofluorescein diacetate assay. The cytotoxicity and haemolytic activity were determined in murine fibroblasts (L929), undifferentiated and differentiated murine fibroblasts (3T3-L1 cells) and human erythrocytes using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and haemolysis assays, respectively. The effects on lipid accumulation in differentiated 3T3-L1 adipocytes were evaluated with the Oil Red O assay at concentrations of 10 and 100 _g/mL. In addition, the effects of 10 and 100 _g/mL of each weight loss compound on erythrocyte morphology and fibrin networks were examined using scanning electron microscopy. Neither L-carnitine nor hydroxycitric acid had antioxidant activity, however, only hydroxycitric acid at 500 – 2500 _g/mL protected 3T3-L1 preadipocytes against oxidative damage. Both did not induce oxidative stress. In contrast, conjugated linoleic acid was found to have antioxidant activity at 25 – 250 _g/mL, however this translated into oxidative damage or pro-oxidant effect in 3T3-L1 preadipocytes. Of concern is that conjugated linoleic acid is marketed as a product with antioxidant properties and this effect was not observed using cellular models. No antioxidant or oxidative effects were observed for ephedrine and conjugated linoleic acid, however at 500 _g/mL both weight loss compounds were cytotoxic. All compounds at 10 and 100 _g/mL did not alter lipid levels or reduce lipid accumulation in differentiated adipocytes. Ephedrine, conjugated linoleic acid, L-carnitine and hydroxycitric acid at increasing concentrations following 3, 24 and 48 hour exposure did not cause human erythrocyte haemolysis. Exposure of human whole blood to the weight loss compounds for 30 minutes, did not cause changes to erythrocyte morphology and the structure of the fibrin network that formed. Findings were that ephedrine, conjugated linoleic acid, L-carnitine and hydroxycitric acid does not adversely affect blood haemostasis. High concentrations of each weight loss compound were used and does not necessary represent blood levels following absorption, which would be lower. It can therefore be concluded that in healthy individuals, these weight loss compounds will not adversely affect cellular function although conjugated linoleic acid and ephedrine were cytotoxic at high concentrations. Future studies should focus on the effects of these compounds on different cellular pathways and the effects on blood should be evaluated in obese patients, where these over-the-counter weight loss compounds may have a beneficial ability to reduce oxidative stress and improve blood haemostasis.