L-Carnitine Supplementation to Diet: A New Tool in Treatment of Nonalcoholic Steatohepatitis—A Randomized and Controlled Clinical Trial

Mariano Malaguarnera AP; Maria Pia Gargante MD; Cristina Russo MD; Tijana Antic MD; Marco Vacante MD; Michele Malaguarnera MD; Teresio Avitabile; Giovanni Li Volti AP; Fabio Galvano AP

Disclosures

Am J Gastroenterol 

In This Article

Methods

Study Design

This was a randomized, double-blind, placebo-controlled study. The study was conducted between January 2004 and December 2006, and the study participants were recruited from Cannizzaro Hospital, Catania, Italy.

This study was designed and conducted in compliance with the ethical principles of Good Clinical Practice Guidelines and the Declaration of Helsinki.[16] The study protocol was approved by the research ethics committee of Cannizzaro Hospital, Catania, Italy. Informed consent was obtained from patients before any study procedures were initiated. Eighty patients with a clinical and pathologic diagnosis of NASH were enrolled in the study. Seventy-four patients (40 men and 34 women; age 28–60 years, mean age 47.6 years) were randomly assigned by a computer-generated randomization schedule to receive a 24-week supply of either L-carnitine or placebo. Thirty-eight patients were allocated to placebo group and 36 were allocated to L-carnitine group. None of the patients withdrew from the planned treatment (Figure 1). The treatment period was 24 weeks. The measurements were made every month, both for efficacy tests and tolerability.

Figure 1.

Trial profile of L-carnitine treatment. NASH, nonalcoholic steatohepatitis.

Patients

Laboratory features of controls and patients included in this study are similar and they are summarized in Table 1 and Table 2 . The data included subjects who had, for at least 6 months, abnormal serum aminotransferase levels that were not related to other causes of liver disease. All patients underwent percutaneous liver biopsy, and the diagnosis of NASH was established by the presence of pericentral macrovesicular steatosis, ballooning degeneration of the liver cells with or without Mallory bodies or fibrosis, and lobular and portal inflammation in the absence of other causes of liver disease (viral, drugs, toxin, autoimmune, metabolic). Significant alcohol consumption was a criteria of exclusion (>10 g per day for females and >20 g per day for males). Other causes of exclusion were hereditary hemochromatosis, α-1 antitrypsin deficiency, Wilson's disease prior surgical procedures such as jejunoileal or jejunocolic bypass, gastroplast, total parenteral nutrition in the past 6 months, pregnancy, use of drugs such as calcium channel blockers, high dose of synthetic estrogens, methotrexate, amiodarone steroids, chloroquine, a history of treatment with lipid-lowering agents, a history of hypothyroidism, or Cushing syndrome. After a 4-week washout period, 74 patients were asked to follow the National Cholesterol Education Program Adult Treatment Panel III therapeutic lifestyle-change diet; then eligible patients were randomized to receive L-carnitine or placebo.[17] A group received L-carnitine 2 g per day divided into two equal doses of one 1 g tablet after breakfast and one 1 g tablet after dinner for 24 weeks (L-carnitine, Sigma Tau, Pomezia, Italy). The other group received placebo according to the same regimen and for the same duration.

Prerandomization Phase

The subjects were required to document all caloric intake with the use of a diary, completed every 2 days. This prerandomization period was designed to nullify the effects of dietary changes on metabolic markers. During the initial 4-week phase, subjects were instructed by a dietitian to follow an ad libitum diet as classified by the National Cholesterol Education Program.[17]

Patients were checked by a dietician every month; at each visit, the dietician provided instructions on dietary intake recording procedures as part of a behavior-modification program, and the patients' resulting food diaries were later used for counseling. All patients in both the groups were given the same 1,600-calorie diet and were prescribed an exercise plan. Both groups had a 30-min home-based whole-body stretching routine to perform three times per week. Subjects received one supervised stretching session at treatment initiation, a booklet detailing the stretches, and were unsupervised thereafter. All individuals were informed that the research hypothesis was that regular stretching could reduce inflammation and assist in the preferential reduction of adiposity from the liver and viscera. Subjects underwent weekly visits throughout the treatment period to assess the adherence to the study protocol, to measure blood pressure, and to record adverse events.

Randomization Phase

Throughout the trial, L-carnitine was supplied in vials with 2 g carnitine taken orally twice a day. All drugs and placebos were identical in appearance, and neither investigators nor patients were informed of the selected agent until the end of the study phase. Dosing instructions were provided with each patient pack. All trial medications were instructed to be taken as prescribed. Subjects were considered compliant if the number of returned vials was between 80% and 120% of the planned treatment regimen. For the duration of the trial, any concomitant drug was administered at the lowest possible therapeutic dosage and, as far as possible, was not changed.

Efficacy Assessment

Throughout the randomization phase of the study, thrice-weekly alimentary diary cards were used to collect efficacy data. The primary efficacy measures were changes in aspartate aminotransferase, alanine aminotransferase (ALT), γ-glutamyl-transpeptidase (γ-GT), albumin, total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, insulin, C-peptide, C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), alkaline phosphatase, and prothrombin time. Measurements were made at the beginning and at the end of the study period. Data were collected in the morning, after an overnight fast.

Tolerability Assessment

Liver biopsy was made at the beginning and then repeated at the end of the treatment period after providing explicit ethics committee approval. In fact, liver biopsy samples remain the only way to establish a definitive diagnosis of NASH and determine the stage of hepatic fibrosis, thereby also providing prognostic information. Body mass index was calculated as weight/height2 (kg/m2). Laboratory assessments were monitored at baseline and monthly, until the end of the trial. These data included hemochrome, glycemia, creatininemia, and blood urea. After providing informed consent, each subject in the two groups underwent ultrasonography examination of the liver. Electrocardiogram and blood pressure were monitored with the use of standard techniques.

Clinical Laboratory Tests

Blood samples were obtained after the patients had fasted for 12 h overnight. Venous blood samples were taken from all patients between 8 AM and 10 AM. Plasma was obtained from the blood samples by the addition of ethylene diaminetetraacetic acid and centrifugation at 3,000 g for 15 min at 4 °C. Immediately after centrifugation, the plasma samples were frozen and stored at –80 °C. The fasting plasma glucose levels were assayed using the glucose-oxidase method with intra- and inter-assay coefficients of variation (CV) of 0.8% and 2.4%, respectively. The total cholesterol and triglycerides were determined using fully enzymatic techniques on a clinical chemistry analyzer whose intra- and inter-assay CVs were 1.2% and 2.3%, respectively, for the total cholesterol measurement and 1.1% and 2.4%, respectively, for the TG measurement. The HDL cholesterol level was measured after precipitation of plasma apo-B containing lipoproteins with phosphotungstic acid. The intra- and inter-assay CVs were 1.0% and 2.0%, respectively. The LDL cholesterol level was calculated using the Friedewald formula.[18] Insulin was measured using a two-site immunoenzymatic assay performed on the Access automated immunoassay system. Intra-assay CVs are 2.1% at 6.70 μU/ml and 2.5% at 116 μU/ml. Inter-assay CVs are 3.8% at 12.7 μU/ml, 4.1% at 48.8 μU/ml, and 4.5% at 121 μU/ml. C-peptide was measured by a direct, double antibody sequential radioimmunoassay. Inter-assay CVs are 4.9% at 0.43 and 1.75% at 4.36 nmol/l. A measure of insulin resistance was performed with homeostasis model assessment (HOMA). The homeostatic model assessment (HOMA-IR) was calculated using the formula IR=insulin × glucose/22.5. Higher values of HOMA-IR indicate more insulin resistance. Serum-sensitive CRP was measured at baseline and halfway through the intervention by a particle-enhanced immunoturbidimetric assay (Roche Diagnostics, Mannheim, Germany). The serum TNF-α was analyzed with the BD Cytometric-Bead Array Kid (BD Biosciences, San Diego, CA) at baseline and at the end of intervention. Samples were processed in duplicate. Laboratory evaluation included serum liver tests (total protein, albumin, aspartate transaminase, alanine transaminase (ALT), γ-GT, alkaline phosphatase, and total serum bilirubin), hepatitis B serology (hepatitis B surface antigen, and antibody to hepatitis B surface antigen, antibody to hepatitis B core antigen), antibody to hepatitis C virus, hepatitis C RNA polymerase chain reaction, autoantibodies (antinuclear antibody antismooth muscle antibody, and antimitochondrial antibody), and iron profile (serum iron, transferring saturation, and ferritin). All values were determined following a 12-h fasting period by the hospital clinical laboratory.

Ultrasonography Test

To perform ultrasonographic scan, we used a real-time machine (Logiq 500, General Electrics) with a linear 3.5-MHz transducer (Pie Medical Scanner 150). This technique is reported to have a high sensitivity and specificity for the diagnosis of fatty liver, defined as the presence of fat in >30% of each hepatic lobule, when a combination of the following four parameters are used: (1) diffuse hyperechoic echotexture (bright liver), (2) increased liver echotexture compared with the kidneys, (3) vascular blurring, and (4) deep attenuation.

Histological Analysis

Liver biopsies were obtained using a 16-gauge Klatskin needle. A liver specimen of 15 mm with at least 10 portal tracts was considered adequate for evaluation. After completion of the study, all liver biopsy samples were coded and read by a hepatic pathologist without the knowledge of the patient or the sequence of the biopsy. Six histological features of NASH were scored semiquantitatively from 0 to 4, including steatosis, acinar zone 3 hepatocellular injury (ballooning degeneration), parenchymal inflammation, portal inflammation, perisinusoidal fibrosis, and Mallory bodies. Ubiquitin immunostaining was used to help identify Mallory bodies. The primary outcome measure for this study was improvement in liver histology as assessed by the NASH-activity index. The NASH-activity index was defined by the sum of scores for steatosis, parenchymal inflammation, and hepatocellular injury, and thus ranged from 0 to 12. Improvement was defined as a decrease in the NASH-activity index of at least three points with improvements of at least one point for each of the three features.[19]

Statistical Analysis

The data were analyzed using the Statistical Analysis System software version 6.11 (SAS Institute, Cary, NC). The differences between the means were evaluated by using analysis of variance or paired t-tests, where appropriate. All results were expressed as mean±s.d. unless otherwise mentioned. Pearson's correlation coefficient was used for correlation analysis between variables. P values <0.05 were considered significant. Tukey's post hoc tests were used to assess the differences between the treatment groups. Data were further analyzed with a Bonferroni adjusted t-test for multiple comparisons. The Mann–Whitney U-test was used to compare nonparametric data. The primary population for statistical analysis was an intention-to-treat population of all randomly assigned subjects.

Comments

3090D553-9492-4563-8681-AD288FA52ACE
Comments on Medscape are moderated and should be professional in tone and on topic. You must declare any conflicts of interest related to your comments and responses. Please see our Commenting Guide for further information. We reserve the right to remove posts at our sole discretion.

processing....