L-Carnitine Supplementation could improve Clinical Symptom but not effect on C- reactive Protein and Malondialdehyde in Obese Women with Knee Osteoarthritis: A Double Blind Randomized Controlled Trial

L-carnitine decreases oxidation and inammation by reducing the fatty acid in plasma and using oxygen in ATP synthesis. So, knee osteoarthritis (KOA) can be improved by reducing apoptotic chondrocytes. The aim of this study was to compare the effect of L-carnitine supplementation along with low calorie diet and low calorie diet alone on improvement of KOA among obese women. In other words, we aimed to investigate the additional effect of L- carnitine on improvement of KOA among obese women who received low calorie diet. improvements compared compared 0.024)); 6. 56 BMI compared 0.027)).

L-carnitine (4-N-trimethylammonium-3-hydroxybutyric acid) plays an important role in transferring the longchain fatty acids from the inner mitochondrial membrane to the peripheral tissues [9]. L-carnitine, by reducing the fatty acid in plasma and using oxygen in synthesis of ATP, can decrease oxidation and in ammation [8].
So, by reduction of apoptotic chondrocytes in OA cartilage, KOA can be improved [10]. Some studies showed decrease of carnitine concentration in blood and tissues in the rheumatoid arthritis [11]. Animal study also demonstrated that L-carnitine can reduce KOA symptoms [12]. Earlier studies showed that L-carnitine can signi cantly reduce CRP and MDA in coronary [29,30] and hemodialysis patients [31]. Meta-analysis also showed that L-carnitine supplementation signi cantly reduced the levels of CRP [13], cholesterol and lowdensity lipoprotein cholesterol (LDL-C) in diabetic patients [14]. Recent study also reported that 750 mg of Lcarnitine supplementation had no effect on serum lipid pro le and hs-CRP in women with KOA [15,16].
However, a study on hemodialysis patients reported no signi cant effect on oxidative stress [32]. The results of studies over the effect of L-carnitine on lipid pro le, CRP, and some other oxidative indices are controversial. Furthermore, rare studies evaluated the effect of this supplementation on KOA. So, we targeted at evaluating the effect of oral L-carnitine supplementation on CRP, MDA, lipid pro le, WOMAC index, as well as anthropometry and body composition measures in obese women with KOA.
Moreover, it was con rmed that low calorie diet had a signi cant effect on KOA [17,24] [17]. Therefore, it seems that both L-carnitine supplementation and weight loss diet are bene cial for OA, but previous studies were conducted on the effect of L-carnitine supplementation and did not include the received calories. The aim of this study was to compare the effect of L-carnitine supplementation along with low calorie diet and low-calorie diet alone on improvement of KOA among obese women. In other words, we aimed to investigate the additional effect of L-carnitine on improvement of KOA among obese women who received low calorie diet.

Study participant
This double blind parallel randomized controlled trial was conducted according to the CONSORT guidelines.
Participants included 76 KOA women recruited from the Khatam Al-Anbia Super Specialty clinic at the Department of Rheumatology, Yazd, Iran. To be eligible for inclusion, women had to be over 45 years old, have a body mass index (BMI) in the range of 25-35 kg/m 2 , and have diagnosed KOA according to the clinical classi cation of KOA (18). The exclusion criteria were: having former or planned knee-joint replacement, being under pharmacologic treatment for obesity, having any history or active presence of other rheumatic diseases, using any nonsteroidal anti-in ammatory drug (NSAID), consuming ≤ 20 percent of the supplements, consuming multivitamin, minerals, or other nutritional supplements, and having severe liver, kidney, or heart diseases.

Randomization and intervention
This study was a double blind randomized controlled trial. Patients, who met the study criteria, were assigned to L-carnitine group (LCG) or placebo group (PLG) through randomization lists made by a computerized random-number generator and simple randomization process in a ratio of 1:1. The L-carnitine group received 1 g/d L-carnitine daily for 12 weeks. The placebo group received a placebo according to the same regimen and the same duration. The placebo pills contained inactive ingredients with no therapeutic activity and had an identical appearance. These tablets were produced by Karen Pharmaceutical & Nutrilife Co., Yazd, Iran. As a double-blind study, the bottles were labeled A and B respectively for the placebo and drug by the factory but neither the patients nor the research team were aware of the codes. Every month, patients received a bottle of tablet containing 30 tablets. Compliance with the medication was monitored by the research personnel using pill counts and patients' self-reporting. Participants who did not consume more than 30 percent of their supplements were eliminated from the analysis. All participants followed a low-calorie diet. A registered dietitian estimated the energy expenditure for each patient through Harris-Benedict formula using the individual activity factor [19]. The recommended composition of the dietary regimen was 50% to 60% carbohydrates, 15% to 20% proteins, and less than 30% total fat. Initially, a dietitian completed the 3-day food recall for all participants and visited patients every month to check their compliance with the diet according to the patients' feedback and 24-hour food recall. At the baseline, physical activity during the past week was assessed using the long version of International Physical Activity Questionnaire (IPAQ). Patients were also asked not to change their level of activity during the study.

Outcome Measurements
The following variables were assessed at the baseline and 12 weeks after the start of treatment: primary outcome included WOMAC, CRP and MDA. Secondary outcome was LDL-C, total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C), triglycerides (TG), BMI, fat mass, free fat mass, as well as waist and hip circumference.
In order to conduct the laboratory tests, 5 mL of venous blood samples was obtained after the patients had fasted for 8 hours overnight. Immediately after the centrifugation (3000g, 10 min), serum samples were produced from the collected blood samples. They were then frozen at -20 ºC, stored at -70 ºC, and measured at the same time. The total TC, HDL-C, and TG were later measured using Pars Ammon kit (Iran). LDL-C was then calculated using Friedewald's equation (20). Serum CRP and serum MDA concentrations were measured through enzyme-linked immunosorbent assay kits and thiobarbituric acid reactive substances Zellbio kit (Germany), respectively.
The patients' weights were also recorded on a portable digital scale (Omeron BF511, Japan) to the nearest 0.1 kg. Participants were in light clothes and stood on the scale without help. Fat mass and free fat mass were measured with this scale. Further, height was measured in standing position using an audiometer xed on a straight wall to the nearest 0.1 cm. Waist circumference (WC) was recorded to the nearest 1 centimeter using non-stretch plastic tape placed midway between iliac crest and lowest rib while participants were in standing position. Hip circumference was also measured over the largest part of buttocks with the accuracy of 1 cm.
BMI was also calculated as weight (kg) divided by height squared (m 2 ). To assess the clinical symptoms Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) were used . Patients lled out the Persian version of WOMAC index [21] that consists of 24 questions (pain, stiffness, and physical functional).
Items should be answered on a Likert scale: none (0), mild (1), moderate (2), severe (3), or extreme (4). For physical activity, the Persian version of IPAQ was applied [22]. The continuous score shows the weekly energy expenditure expressed in MET-min/week (metabolic equivalent-minutes). Individuals were classi ed into three categories of 'inactive', 'moderately active', and 'highly active' using the categorical classi cation.

Sample size
Power calculations were conducted based on pain scores of 72 women with KOA who participated in a previous trial [23]. On the basis of an assumed 10% dropout rate, we estimated that a total sample size of 76 patients (38 patients per group) would provide 80% of the power to detect a 2.6 pain score difference between the PLG and LCG.

Statistical analysis
Statistical analyses were carried out using SPSS (version16). The normal distribution of variables was tested by Kolmogorov-Smirnov test. Differences in patients' anthropometrics, WOMAC scores, and hematological measurement data between the PLG and LCG were analyzed by Student's t-test or the Mann-Whitney rank sum test for parametric and non-parametric continuous variables, respectively. The paired t-test or Wilcoxon signed rank test was used to analyze the data within each group before (baseline) and after the intervention (week 12). Analysis of covariance (ANCOVA) was used to identify the differences between the two groups after adjusting for change weight. Results were considered statistically signi cant at P < 0.05. Normal data were indicated by means ± standard deviations (SD) and the non-normal scores were presented in median and inter quartile range (IQR). The dietary information was analyzed with N4 software (Nutritionist: version 4.0; Tinuviel Software, Warrington, United Kingdom).

E cacy and tolerability assessment
For the safety, all participants were interviewed every month for any signs of L-carnitine toxicity or other adverse problems considering the diet, including serious illnesses or hospitalizations.

Study participant characteristics
The sampling and trial pro les are summarized in Figure 1. The baseline characteristics of these patients are shown in Table 1. Participants included 76 women with a mean age of 54.73 ± 7.41, BMI of 32.65 ± 5.60 kg/m 2 , and percentage of body fat of 44.42 ± 5.90%. Median and IQR baseline of CRP was 3.52 ± 4.33 mg/dL. Anthropometric, lipid pro le, CRP measures, physical activity, and diet composition such as total energy, protein, fat, and carbohydrate intake, as well as education and occupational status did not differ between groups at the baseline of the study (P > 0.05).

Blood lipids and lipoproteins, CRP and MDA
No signi cant decrease was observed in the LDL-C, HDL-C, TG and CRP concentrations in either the LCG or PLG compared with the baseline. In the LCG, however, there was a signi cant decrease in TC (226.74 ± 50.55 to 212.24 ± 41.39 mg/dL, P= 0.021) and MDA (33.32 ± 36.09 to 26.14 ± 22.22 mg/dL, P= 0.035) after treatment but not in PLG. No signi cant difference was observed between the LCG and the PLG in lipid pro les, CRP, and MDA concentrations ( Table 2).

Anthropometry and body composition
Compared with the baseline, weight, BMI, as well as the waist and hip circumference decreased signi cantly in both groups after 12 weeks of intervention (P= 0.001) ( Table 3). Furthermore, results showed signi cant reduction in visceral fat and fat mass in LCG (P = 0.001), but not in PLG. However, no signi cant differences were observed between the LCG and the PLG. The mean of weight change was 4.49% in the LCG, while it was 2.43% in the PLG (P = 0.05). changes of BMI were signi cant between the two groups (-1.21 ± 0.84 kg/m 2 compared with -0.79 ± 0.70 kg/m 2 ; P = 0.027).

WOMAC index
Compared with the baseline results, decrease of pain, stiffness, physical function, and total scores were signi cant in both groups after 12 weeks of treatment (P= 0.001). Physical function improved signi cantly (P= 0.014) in the LCG with a mean of 11.15 ± 6.56 compared with PLG with a mean of 15.6 ± 8.2.
Furthermore, the patients in the LCG had signi cantly lower total scores (17.41± 9.81 versus 23.50 ± 12.02) than those in the PLG (P= 0.024). However, mean of changes were not signi cant ( Table 2).

Tolerability
Both L-carnitine and placebo were well tolerated in all patients. In the group treated with L-carnitine, one patient complained of skin dryness and two complained of slight stomachache. In the PLG, two patients complained of skin dryness and three complained of stomachache.

Discussion
This randomized placebo-controlled trial examined the effect of oral l-carnitine (1000 mg/d) consumption compared with placebo in combination with low calorie diet for 12 weeks in overweight or obese women with KOA. Results showed signi cant improvements in physical function, total score WOMAC, BMI, and weight.
However, MDA, total cholesterol, fat and visceral fat mass decreased only in LCG signi cantly, but not in the PLG. CRP and other lipid pro les did not change signi cantly.
One of the important risk factors of KOA is obesity. Studies con rmed that weight loss intervention can improve pain and physical function [17,24]. Therefore, it seems that both L-carnitine supplementation and weight loss diet are bene cial for OA. To the best of our knowledge, this is the rst study investigating the effect of L-carnitine supplementation along with low calorie diet compare low calorie diet alone on improvement of KOA among obese women.
This study showed a signi cant decrease in BMI and weight in the LCG. In agreement with our ndings, a meta-analysis reported that L-carnitine might be effective for weight loss in adults [25]. Another study that supported this hypothesis indicated that L-carnitine can decrease weight, BMI, as well as waist and hip circumference [26]. In other words, studies assessing the effect of L-carnitine on other diseases didn't report signi cant effects on anthropometry measures [14,27]. In our study fat mass and visceral fat mass were not differences signi cantly between two groups. Similar to our results, no signi cant change was observed in fat mass of healthy people who consumed L-carnitine with exercise for 8 weeks [27]. On the contrary, 2000 mg Lcarnitine among with hypo-caloric diet could reduce fat mass in diabetic patients [28]. The doses of Lcarnitine supplementation in the mentioned study were two times higher than those of our study that might explain the discrepancy between results. L-carnitine leads to weight loss by oxidizing fat and decreasing the serum levels of leptin. The serum level of leptin is proportional to the body fat mass. Thus, the level of leptin drops by decrease of the adipose tissue mass [29]. Moreover, obesity is a chronic in ammatory disease that causes lipid peroxidation by abnormal production of pro-in ammatory factors such as IL-6 and CRP as well as release of free fatty acids from adipose tissue [4]. This study concluded that, no signi cant differences were observed in CRP and MDA between the LCG and the PLG. In consistent with our results, 750 mg/d of Lcarnitine supplementation on women with KOA didn't show any signi cant change on MDA and hs-CRP [15,16]. Con rming this issue, supplementation with oral consumption of 1000 mg/d L-carnitine can signi cantly reduce CRP and MDA levels in coronary patients [30,31]. These results were similar to another study in which propionil L-carnitine was injected into hemodialysis patients [32]. Furthermore, a study on hemodialysis patients with hyper lipo-proteinemia reported that 1000 mg/d of oral L-carnitine could reduce in ammation but didn't affect oxidative stress [33]. Moreover, a meta-analysis con rmed that L-carnitine can reduce CRP [13]. In comparison with the current research, CRP reduction in this study may be due to injection of L-carnitine and longer duration of the study.
Our results, which are consistent with the literature [15,34], showed that oral administration of L-carnitine didn't lead to any signi cant difference in lipid pro le. In consistent with the present ndings, two metaanalyses indicated that L-carnitine decreases serum LDL-C [35] and cholesterol [14]. L-carnitine is an essential cofactor that helps transfer of fatty acids into mitochondria and causes incorporation of long-chain fatty acids into the β oxidation cycle to produce acetyl-CoA. L-carnitine helps oxygen entrance into the tri carboxylic acid (TCA) cycle to synthesize ATP. This process decreases the concentration of oxygen and reduces formation of reactive oxygen species [36].
However, our study showed signi cant improvement in physical function and total score in WOMAC questioner. In the same line with our results, one study indicated 750 mg of oral L-carnitine supplementation have signi cant improvement in pain intensity and patients' global assessment of disease status on KOA [23]. A review article indicate that more than 2 g L-carnitine can reduce stiffness in movement, the pain after prolonged movement, and disturbed sleep due to the pain [37]. L-carnitine may have an additional effect on the improvement of KOA. In other word, L-carnitine may have a greater impact on the clinical symptoms of KOA by decreasing the fat mass and causing a signi cant reduction in WOMAC score. It seems this effect are independent of in ammatory and lipid pro le modi cation [15,23]. After adjusting for the weight reduction during the study, the results remained signi cant, which indicates that these effects were independent of weight change. This may show the direct effect of the L-carnitine.
The strength of our study was comparing the effect of L-carnitine supplementation along with low calorie diet on improvement of KOA among obese women for the rst time; their adherence to the diet was also monitored every month. Moreover, L-carnitine appeared to be well tolerated by participants. The limitation of the present study was that we did not evaluate the serum L-carnitine levels. Long-term studies with higher doses of L-carnitine and measurement of other indicators such as in ammation factors and MMP would help to clarify further this realm.

Conclusions
According to the ndings, Oral administration of 1000 mg L-carnitine in 12 weeks could improve WOMAC score and BMI, but not affect CRP, MDA, and lipid pro le. Further trials with higher doses and longer durations, are required to verify our results.   2 Determined with the use of paired Student's t tests for differences between baseline and follow-up in the L-carnitine group 3 Determined with the use of paired Student's t tests for differences between baseline and follow-up in the placebo group.
4 Determined with the use of independent samples t tests between L-carnitine and placebo groups. 1 Determined with the use of paired Student's t tests for differences between baseline and follow-up in the L-carnitine group 2 Determined with the use of paired Student's t tests for differences between baseline and follow-up in the placebo group.
3 Determined with the use of independent samples t tests between L-carnitine and placebo groups