The Effect of L-Carnitine on Wound Healing by Secondary Intention in an Animal Model

F ull-thickness skin defects consist of loss of the entire epidermis and underlying dermis. This type of wound tends to heal by secondary intention in which contraction and angiogenesis, in addition to epithelization, gain importance. In this type of healing, inflammatory reaction is more intense, and larger amounts of granulation tissue form compared with healing by primary intention. The increased contribution of myofibroblasts supports the contraction that takes place in this healing process. Additionally, circulatory and metabolic status is important in influencing wound closure.¹ L-carnitine [(CH₃)₃-N-CH₂-CHOH-CH₂-COO-] is an endogenous cofactor in the shuttle mechanism to transport long-chain fatty acids into the mitochondria where they undergo ß-oxidation.² Through the reaction hydrolyzed by carnitine acetyltransferase, carnitine produces free coenzyme A (CoA) for other metabolic reactions and reduces the ratio of acetylCoA to CoA, which stimulates pyruvate dehydrogenase to enhance oxidative use of glucose, reducing lactate production and acidosis. L-carnitine is essential in providing efficient regulation of energy flow from different oxidative sources.^3,4 During ischemia, this regulation is not possible. Excess acetylCoA esters accumulate, a larger amount of carnitine is required to remove them, and secondary carnitine deficiency develops. Tissue carnitine is progressively lost as the ischemic process advances.⁵ L-carnitine or its derivatives have been used in the treatment of angina pectoris, acute myocardial infarction, postmyocardial infarction, congestive heart failure, peripheral vascular disease, dyslipidemia, diabetes, and chronic renal diseases. Satisfactory improvements have been achieved in cellular function and organ performance.^6–10 Moreover, it was shown previously that carnitine had a significant dose-dependent effect to promote random pattern skin flap survival.¹¹ To the authors’ knowledge, this is the first study that attempts to elucidate any possible positive effect of L-carnitine on healing of full-thickness skin defects in rats. Material and Methods Twenty-two Sprague-Dawley rats weighing 200–250g were used in this study. The rats were anesthetized with intraperitoneal ketamine 10mg/100g injections. After scrubbing the dorsal area with 10% solution of povidone iodine to decrease the risk of infection, a 4-cm diameter circle was drawn with a marker. A full-thickness circular skin graft with a surface area of 12.56cm² was harvested from the dorsum of each rat using surgical scissors. The full-thickness skin defect was left to heal by secondary intention, and each rat was kept in a separate cage with the defect uncovered (Figure 1). The rats were divided into 2 groups of 11 rats. The first group was the study group and was given 100mg/kg/day of L-carnitine via intraperitoneal injections (CARNITENE 1g ampoule, sigma-tau Industrie Farmaceutiche) for 10 days. After weighing each rat (rats were 200–250g in weight with a medium of 221g) separately, the dose was calculated accordingly and applied with an insulin injector. The L-carnitine dose was adjusted according to human daily dose, as there is no prescribed dose for rats to date.^11–13 The intraperitoneal route was chosen because of its accuracy and ease of application in rats. The second group was the control group and was given 0.09g/day of sodium chloride as saline solution (0.9% NaCl) intraperitoneally. Both groups were fed a standard laboratory diet and tap water throughout the study. On the 11th day, after removing the crusts—the cover of inflammatory remnants over the wounds, not the epithelized areas—with a surgical instrument, the defect on each rat was outlined on a transparent plastic sheet, and the area of the defect was measured using planimetry. The area was expressed in cm². Results Because the authors’ institute has not yet developed a formal ethics committee for animal experiments, the authors followed the principles outlined in the revised Declaration of Helsinki (1989). During the study, the authors did not observe any wound infection or mortality. Crust formation was observed, and the crusts were removed before the time of measurement to facilitate examination of the unhealed defect areas. The unhealed defect areas of the 2 groups measured on the 11th day were statistically compared using Mann Whitney U test. The means of the unhealed areas were 2.84cm² (95% confidence interval [CI] 1.60cm²; 4.09cm²) and 3.43cm² (95% CI 2.58cm²; 4.29cm²) for L-carnitine and control groups, respectively. The median of the unhealed areas of the study group was 2.7cm² (95% CI 0.97cm²; 4.25cm²), whereas that of the control group was 3.4cm² (95% CI 2.8cm²; 4.5cm²) with a standard deviation of 1.85cm² (95% CI 1.29cm²; 3.25cm²) and 1.28cm² (95% CI 0.89cm²; 2.24cm²), respectively (Figure 2A). There were no statistical differences between the areas of the defects of the 2 groups at significance levels 0.05 and 0.1. The calculated p value was 0.33. Figure 2B demonstrates the areas of the defects of the 2 groups measured at the end of the study. Discussion Various mechanisms of action of carnitine on ischemic tissue have been proposed. Carnitine activates the transport of adenine nucleotides across the inner mitochondrial membrane by preventing adenylate translocase inhibition by long-chain fatty acid accumulation, resulting in increased adenosine triphosphate (ATP) concentration in the tissues, reducing cellular injury, and protecting cells from subsequent episodes of ischemia. The more severe the ischemic disease, the greater the amount of carnitine required to remove the accumulation of acetylCoA esters. Through this process, carnitine improves circulatory reserve of the ischemic tissue. It might permit the ischemic tissue to utilize its remaining limited oxygen supply more efficiently by improving its functional circulatory reserve.^14,15 There is an abundance of literature regarding carnitine and its use in ischemic conditions. Pepine¹⁴ indicated that carnitine may have potential in the management of both chronic and acute ischemic conditions, where it can possibly moderate the tissue damage induced by ischemia. Corsi et al.¹⁶ determined that L-propionyl-carnitine (a derivative of L-carnitine) improves circulatory reserve of the ischemic tissue and has no effect on heart rate and arterial blood pressure and that its effect is possibly due to a drug-induced increase of ATP utilization by the ischemic tissues. According to Pola et al.,¹⁷ carnitine is able to correct tissue hypoxia by increasing ATP and energy production and has the capacity to prevent alterations in endothelial membrane permeability. L-carnitine is suggested to be effective in peripheral vascular disease. Brevetti et al.¹⁸ found that L-carnitine, although not affecting blood flow and blood pressure under resting conditions, improves the functional circulatory reserve in patients with peripheral vascular disease. Bolognesi et al.¹⁹ indicated that the beneficial effect of propionyl-L-carnitine in peripheral arterial disease is not due to a direct vasoactive action. However, Cevese et al.²⁰ indicated that propionyl-L-carnitine has a direct vasodilator effect in musculocutaneous vascular beds at high doses and probably enhances tissue metabolism. In their study on skin flap survival in rats, Tellioglu et al.¹³ determined significant dose-dependent effect of carnitine to promote random pattern skin flap survival. Peripheral vascular disease in an extremity and a full-thickness skin defect may resemble each other with respect to ischemia in the related area. In the case of a full-thickness skin defect in an extremity, a lack of vascular supply leads to ischemia. To overcome this, a secondary type of healing begins. Epithelization, contraction, and angiogenesis are the processes that take place in this type of healing. These steps need augmentation in glucose consumption. The authors hypothesized that these processes may be enhanced by L-carnitine supplementation, although ulcerative lesions in peripheral arterial diseases may not be the same depths as full-thickness skin defects but similar. L-propionyl-carnitine has been shown to improve the healing process in these lesions.⁹ Additionally, L-carnitine has been suggested to have a specific trophic effect on type 1 human skeletal muscle fibers, which are characterized by oxidative metabolism. This is important, as contraction is a main mechanism in wound healing by secondary intention.²¹ The authors could not locate literature on the effect of carnitine on wound healing by secondary intention. Consequently, the authors hypothesized that carnitine may be effective in secondary wound healing. Oral human dose of L-carnitine ranges between 50–200mg/kg, whereas intravenous dose is 15–140mg/kg. In the present study, an average daily dose was chosen with respect to human daily dose. Intraperitoneal route was chosen, as the dose parallels an intravenous dose, and the variability in gastrointestinal absorption is overcome by use of the intraperitoneal route. For oral routes, dosages of 0.5–4g/day, divided to 2–3, are generally chosen. Systemic bioavailability of oral carnitine is low (5–20%), reflecting intestinal absorption barrier and hepatic first-pass elimination. Oral administration of propionyl-L-carnitine is found to be safe without significant hepatic, renal, metabolic, or gastrointestinal side effects. The majority of L-carnitine is rapidly eliminated by the kidneys.⁵ L-carnitine did not produce any observable side effects in the authors’ study groups. According to the authors’ results, there was no significant effect of L-carnitine on secondary wound healing in rats. However, there was a tendency toward faster healing under the effect of L-carnitine, though insignificant. On gross examination, the healing areas of the L-carnitine-treated group appeared to be more mature, unlike the control group, which had a tendency to bleed easily; however, this observation needs further objective methods for evaluation. The defects were not occluded by any means to overcome any possible effects of these materials. The dose, administration, and duration of the drug treatment may not be appropriate, resulting in lack of significant difference between the 2 groups. The selection of the animal species and type of the defect for this experiment may also be controversial, as rodents tend to heal mostly by contraction. A pig or sheep model may be more suitable. However, the authors’ laboratory facilities are limited for using different species of animals. Studies are planned using a higher dose, direct or intraperitoneal administration, and longer duration of L-carnitine treatment. Conclusion L-carnitine has been effective in various ischemic conditions. However, it has been shown not to significantly promote secondary wound healing in a rat model, although there was a tendency toward faster healing in the group receiving the drug. Further studies using different animal models, L-carnitine dosages, and durations of treatment are necessary to elucidate any possible effect. Acknowledgement The authors thank Ömer Bozdogan, PhD, Professor, and Eylem Suveren Tiryaki, MSD, from the Department of Biology, Abant Izzet Baysal University, and Ms. Neslim Hancilar for their technical assistance.