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Journal of the American College of Nutrition, Vol. 24, No. 3, 172-176 (2005)
Published by the American College of Nutrition

Decreasing Oxidative Stress with Choline and Carnitine in Women

Dileep S. Sachan, PhD, FACN, Nobuko Hongu, PhD and Maike Johnsen, MS

Department of Nutrition and Agricultural Experiment Station, University of Tennessee, Knoxville, Tennessee

Address reprint requests to: Dileep S. Sachan, DVM, PhD, Professor, Nutrition Department, 229 JHB, University of Tennessee, Knoxville, TN 37996-1920. E-mail: dsachan{at}utk.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSION
 REFERENCES
 
Objective: Fatty acid oxidation is predominantly a mitochondrial event, which is enhanced by dietary choline and carnitine supplementation resulting in extra reactive oxygen species (ROS) load. The objective was to assess oxidative stress level by thiobarbituric acid reactive substances [TBARS] in choline and carnitine supplemented healthy women before and after mild exercise.

Methods: Nineteen free-living women completed the placebo control study in which choline and/or L-carnitine was orally taken for 21 days. Anthropometric measurements, dietary recall, exercise routine and blood samples were analyzed to determine body composition, nutrients intake, distance walked and biochemical markers related to oxidative stress.

Results: TBARS were significantly lower in the groups supplemented with choline, carnitine or both and the mild exercise (walking) was not a deterrent in this effect of the supplements. Serum vitamin A and E concentrations were higher in the supplemented groups even though the consumption of these nutrients was not different among the groups.

Conclusion: Choline and carnitine supplementation lowers lipid peroxidation, and promotes conservation of retinol and {alpha}-tocopherol in free-living women.

Key words: antioxidant, oxidative stress, choline, carnitine, women


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 CONCLUSION
 REFERENCES
 
Oxidative stress being associated with many chronic diseases has lead to expansion of the list of antioxidants as dietary supplements. The main cause of oxidative stress is excessive production of ROS that alter macromolecules such as lipids, proteins and nucleic acids with concomitant change in structure and function of cells and organs [1]. Most studies of oxidative stress have been conducted in animal models for lack of better invasive techniques in humans. The few studies in humans are limited to male adults; and there is paucity of data on aged, children, and women [2].

Earlier we have shown that choline promotes carnitine conservation in humans and animals and this conservation is reflected in accretion of carnitine by all tissues especially the skeletal muscle [3,4]. A functional consequence of the carnitine accretion was the loss of body fat, enhanced fatty acid oxidation and disposal of acylcarnitines in urine without affecting respiratory exchange ratio (RER) in animal models [5–7]. The fatty acid mobilization, oxidation and excretion were also promoted by choline and carnitine supplementation and exercise in women [8]. Fatty acid oxidation is predominantly a mitochondrial event and it is enhanced by starvation, exercise and diet supplementation [9]. Mitochondrial machinery that oxidizes energy substrates and carries out respiration also produces significant amounts of ROS, which leak out of mitochondria and cause substantial damage to various cellular components [10]. Enhanced mitochondrial activity is expected to enlarge the ROS pool and, in turn, promote oxidative stress [11]. Choline and carnitine supplementation indicated enhanced mitochondrial activity [8]. Therefore, we set out to determine a marker of oxidative stress in women supplemented with choline and carnitine along with mild exercise regimen for weight loss.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 CONCLUSION
 REFERENCES
 
Subjects and Study Design
The protocol of this study was approved by the Institutional Review Board for the Protection of Human Subjects in Research. Participants varied in age from 18–54 y, body weight 47.5–92.7 kg, BMI 18.9–35.9 kg/m2, % body fat 17.9–37.8 and waist to hip ratio (WHR) 0.71–0.89 (Table 1). Those who met the inclusion criteria were informed about the nature and purpose of the study and were required to submit a written informed consent. A randomized placebo-control study was designed with 3 groups lasting a period of 21 d during which dietary and exercise interventions were carried out. At baseline, habitual dietary intake was assessed and anthropometric measures were taken. Fasting venous blood samples were collected from each group before starting the treatments (baseline or zero time value) and at the end of each intervention period. All participants were specifically instructed to follow their usual life style, and not to introduce new foods or dietary supplements to their diets [8].


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  		Table 1. Baseline Characteristics of the Subjects in the 3 Groups

 
Treatments
The participants were randomly assigned to the placebo (n = 7) or supplement groups. Supplement groups were randomly divided into two subgroups; supplement 1 (S1) and supplement 2 (S2). The S1 group (n = 6) received only choline supplementation for wk 1 followed by adding carnitine supplement in the wk 2 through wk 3. The S2 group (n = 6) received only carnitine supplement for wk 1 followed by adding choline in the wk 2 and continued through wk 3. Choline was supplied as choline bitartrate (Pharmachem Laboratories, South Hackensack, NJ) and administered at the dosage of 0.94 g or 7.8 mmoles choline/d. Carnitine was L-carnitine L-tartrate (Lonza, Switzerland) supplied by Daily Manufacturing, Inc. (Rockwell, NC) and administered at the dosage of 0.68 g or 4.2 mmoles carnitine/d. The supplements were US Pharmacopeia (USP) grade with purity ≥98.6%. Each supplement was dissolved in distilled water and a week’s supply dispensed in amber-colored bottles with a coded label and dosing instruction. The placebo bottle contained citric acid in equivalent dosage (1 g or 0.2 mmole/d). All participants were asked to take supplements with daily meals, to keep a record of timing on a daily-supplement sheet and to return the sheets and empty bottles on the following visit. All subjects recorded their baseline physical activity using an electronic pedometer (Yamax Digi-Walker SW-701, Yamax Inc., Japan). They recorded daily walking steps and walking distance (estimated from their strides) as well as non-exercise related physical activities such as occupational and household activities. All participants were asked to undertake > 20 min of aerobic activity, 3–5 times/week during the 3rd week and monitor it by a step counter [8].

Assessment of Body Composition and Dietary Intake
Assessments of body composition were made weekly after an overnight fast. Body weight and height were used to calculate BMI. Skinfold thicknesses were measured 3 times to the nearest 0.1 mm with a Lange skinfold caliper (Cambridge Scientific Industries, Inc., Cambridge, Maryland) on the one side of the body at the triceps, subscapular, suprailiac, thigh, and abdomen. Percentage body fat was estimated using the average of 3 readings according to Jackson and Pollock [12]. Also, percentage body fat was estimated by bioelectrical impedance analysis (BIA). Waist and hip circumferences were measured and waist-to-hip ratio (WHR) was then calculated [13]. Dietary intake was assessed at baseline and every wk of the intervention by 3 d dietary records. Each of the five 3 d periods consisted of the 1 weekend day and 2 weekdays to control for day-of-the-week effects. At the start of the study each participant was given food record sheets and taught, by a registered dietitian, how to record dietary intake. Completed weekly diet records were brought to the clinic, reviewed by the dietitian, coded, and analyzed using the Nutritionist IV program (version 4.1, 1997, First Data Bank, San Bruno, CA).

Blood Collection and Analysis
Venous blood samples were collected following overnight fasting at the beginning of baseline (0 d) and at each visit (7, 14, and 21 d). Whole blood was collected in vacutainer tubes without anticoagulants and kept on ice. After clotting, serum was separated by centrifugation and stored at ~80°C until used for determination of triglycerides [14], cholesterol [15], free-fatty acids [16], proteins [17], TBARS [18], retinol and {alpha}-tocopherol [19].

Statistical Analysis
The data in the Tables are expressed as means ± SEM. All data were statistically evaluated for differences among the means using the Personal Computer Statistical Analysis System for Windows (Version 8e, 2000, SAS Institute, Cary, NC). Two-way ANOVA with repeated measures was performed to determine whether there were any significant differences on variables listed in a table. If there was a significant main effect (group, time, interaction of group and time), Duncan’s multiple range test was performed to determine how the groups differed. Statistical significance was set at p < 0.05. The significant differences in the means of different groups at various time periods are marked by superscript letters, however, significant differences between baseline and a time period within a group have also been analyzed and recognized in the discussion of the results. Only one set of superscripts are shown in tables to avoid bulk of superscript letters.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
CONCLUSION
 REFERENCES
 
The anthropometrics, serum lipids, and dietary intakes of the participants at the start (base line) of the study were not significantly different across the groups. However, base line physical activity was significantly lower in the S2 subjects (Table 1). Serum concentrations of retinol and {alpha}-tocopherol were significantly different across the groups on day 14 and 21 but not on day 0 or 7 (Table 2). The relative decreases in the concentration of these antioxidants after 7 days of choline (S1) or carnitine (S2) supplementation (10%) compared to the placebo (20%) were not significant. The combination of choline and carnitine supplementation restored concentrations of retinol and {alpha}-tocopherol in contrast to no such effect in the placebo group. Mild exercise (day 14–21) did not affect these measures.


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  		Table 2. Alterations of Serum Concentrations of Retinol and {alpha}-tocopherol in Placebo and Supplement Groups, Attributed to the Citrate (Placebo: 0–21 d), Choline or Carnitine (7 d), the Combination of Choline and Carnitine (14 d) and Exercise (21 d)

 
Serum concentration of TBARS was not different among the groups at the baseline and it remained essentially unchanged in the placebo group during the 21 day period (Fig. 1). In contrast, serum TBARS concentrations were significantly lower following supplementation with choline and carnitine either alone or in combination and the mild exercise regimen did not alter this outcome. Although the baseline exercise activity (steps) was significantly lower in the group supplement 2, the increments in steps during the exercise intervention period were not significantly different among the three groups (Table 1). Differences in the supplemented groups on day 7, 14 and 21 were not significantly different.



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  		Fig. 1. Serum TBARS (nmol/mg protein) at baseline and after a 1-wk of different treatments in the 3 groups. The values of the treatment groups are significantly lower than those of the placebo (p < 0.05). P = placebo treatment, Ch = choline supplementation, Cne = carnitine supplementation, Ch + Cne = mixture of choline and carnitine supplementation, Ch + Cne + Ex = mixture of choline and carnitine supplementation with exercise treatments.

 

   DISCUSSION
 
The decrease in serum concentrations of TBARS following a short period of supplementation with choline and carnitine indicates that these two metabolically essential nutrients may lessen oxidative stress in humans (Fig. 1). Though there is some concern about the specificity of TBARS as the biomarker of oxidative stress, it is the most widely used marker for it is inexpensive and easy to assay [1,2]. In a recent study the decrease in malondialdehyde was significantly related to a decrease in 8-epi-PGF2{alpha} and LDL hydroperoxides and increase in GSH:GSSG [20]. The lipid peroxidation, in most human studies, has been assessed by measuring TBARS that are a reasonable reflection of malondialdehyde (MDA), a nonlipophilic peroxidation product from nonconjugated polyunsaturated fatty acids (PUFA) such as arachidonic acid [2]. Since there is paucity of data on the oxidative stress in women, our data is expected to fill in some of this void in knowledge.

It may be premature to add choline and carnitine to the list of the established antioxidants [2,21,22], nonetheless, cumulative evidence may not be entirely ignored. For example, carnitine administration, intra-peritoneal (i.p.), has been shown to prevent TBARS formation in cerebral cortex, cerebellum, hypothalamus, hippocampus, and striatum of the aged rats. There was reversal of the age-associated changes in the brains in old rats achieved by the elevation of antioxidants with L-carnitine [23]. There was restoration of age related decrease in the activities of antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase, and non-enzymatic antioxidants such as reduced glutathione, ascorbic acid and tocopherol in the blood and skeletal muscle mitochondria of aged rats [24]. Carnitine treatment (0.6 g/kg, daily, i.p.) for 2 weeks completely normalized levels of plasma cholesterol, triglyceride, free carnitine and TBARS in the streptozotocin-induced diabetic rats. Also, there was restoration of the endothelium-dependent relaxation response to acetylcholine and complete normalization of the oxidant/antioxidant state [25]. Treatment of aged mice with acetyl-L-carnitine (400 mg/kg, i.p.) three times a week up to 4 months of age ameliorated memory performance and reduced brain lipid hydroperoxide concentration [26]. Patients supplemented with carnitine showed reduction in production of ROS associated with increased sperm motility and viability [27]. These observations support the antioxidant role of carnitine, which is most likely due to stabilization of various membranes including that of the mitochondria [28].

The serum retinol and {alpha}-tocopherol concentrations were stabilized by the combination of supplements (d-14) and were not adversely affected by the exercise intervention (d-21). It is difficult to say if it was an effect of duration of supplementation (14 d) or the combination of choline and carnitine (Table 2). It may be noted that the concentration of retinol and {alpha}-tocopherol decreased only 10% after 7 days of choline (S1) or carnitine (S2) supplementation compared to the 20% decrease in the placebo group in the same period of time (Table 2). However, the dietary intake of nutrients including vitamin A and E were not significantly different among the groups (Table 1). Thus retinol and {alpha}-tocopherol sparing effect of choline and carnitine may be related to membrane stabilization effects of these molecules. If the oxidative damage to membranes is minimized, the antioxidant vitamins will be spared. Collectively these observations advance the hypothesis that choline and carnitine favorably modulate oxidative stress most likely by preventing membrane fatty acid peroxidation.

The exercise regimen enhances mitochondrial activity and therefore, enlarges ROS pool [9–11,29]. The benefits of antioxidant vitamins have been demonstrated under the conditions of high intensity exercise [30]. Such an effect was not seen in our subjects who undertook a mild exercise regimen (walking). The concentration of TBARS did not change significantly over the 21 d in the placebo group who underwent exercise regiment similar to that of the supplemented group. We chose a mild exercise regimen because mild to moderate physical activity is practical and does not require high doses of oxygen as in intense and prolonged exercise regimens [2].


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSION
REFERENCES
 
Choline and carnitine supplementation reduced oxidative stress, and the mild exercise regimen was not a deterrent to this outcome in humans. The supplements promoted conservation of retinol and {alpha}-tocopherol.

Received July 26, 2004. Accepted February 2, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSION
 REFERENCES
 

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