A total of 34 subjects diagnosed with an ASD, aged from 3 to 10 yrs-old (30 males, 4 females) were recruited to the study. The study subjects had bodyweights between 13.2 Kg to 40.4 Kg. None of the study subjects had previously received carnitine-based therapy or previous methionine or lysine supplementation. None of the study subjects had any change in therapy or treatment (including medications) within 1 month prior to the study. The study protocol received Institutional Review Board (IRB) approval from Liberty IRB, Inc. (Deland, Florida). All parents signed a consent and Health Insurance Portability and Accountability Act (HIPAA) form and all received a copy.
Childhood autism rating scale (CARS)
Study participants were evaluated using a CARS test conducted only by a single study investigator (JKK) who observed the subjects and interviewed the parent(s), and was unaware as to the treatment status of the subject. The CARS test is a 15-item behavioral rating scale developed to identify autism as well as to quantitatively describe the severity of the disorder. The CARS test is a well-established measure of autism severity [9
]. The internal consistency reliability alpha coefficient is 0.94; the inter-rater reliability correlation coefficient is 0.71; and the test-retest correlation coefficient is 0.88 [10
]. CARS scores have high criterion-related validity when compared to clinical ratings during the same diagnostic sessions, with a significant correlation of 0.84 [10
Autism treatment evaluation checklist (ATEC)
Each study subject was evaluated by their parents using an ATEC form. Parents were unaware as to the treatment status of their child. The ATEC, designed by the Autism Research Institute (San Diego, CA, USA), is a one-page form [11
]. It consists of four subtests designed to measure the effects of treatment in persons with autism. The items are: (1) Speech/Language/Communication (14 items); (2) Sociability (20 items); (3) Sensory/Cognitive Awareness (18 items); and (4) Health/Physical/Behavior (25 items). The internal consistency reliability of the measure is high (0.94 for the Total score). The ATEC has been successfully used to measure treatment effects in autism [12
Modified clinical global impression (CGI)
An overall CGI score was collected by a single study investigator (JKK) unaware of the treatment status of the study subject using a 3 point scoring system defined as follows: subject improved =1, subject the same =2, and subject worse =3.
Hand muscle testing
Each subject had their hand muscle strength tested using a pneumatic, adjustable squeeze pinch-gauge/dynamometer (Baseline Evaluation Instruments; White Plains, NY, USA) by a study investigator unaware of the treatment status of the subject. This instrument is a reliable and valid method for obtaining muscle force or torque measurements in children [15
]. Subjects were tested using the smallest hand grasp bulb, and were given as many tries as needed to register their maximum grasp reading measured in kilopascals (kPa) for each hand. Special emphasis was placed to ensure that the subject positioned the bulb in the palm of the hand and held the bulb in space to ensure that pressure was not applied by the study subject against a fixed surface. In addition, each study subject was strongly encouraged by a study investigator to give maximum effort.
Treatment adherence measure (TAM) form
A treatment adherence measure (TAM) form was completed by the parents of each study subject. Parents were unaware as to the treatment status of their child. The TAM is a ten-item self-report on treatment adherence that asks specific questions regarding the dose and frequency of use. The TAM was used to calculate the level of adherence to the treatment. It is a Morisky-type self-report adherence measure, designed to measure treatment adherence. Morisky-type adherence measures have been used widely, demonstrating good reliability as a self-report measure [17
Frequency and intensity of side effect rating (FISER)/global rating of side effect burden (GRSEB)/patient report of incidence of side effects (PRISE)
The FISER/GRSEB/PRISE forms were completed by the parents of study subjects that were unaware of the treatment status of their children. The FISER/GRSEB/PRISE forms include global measures, each using a 7-point Likert-type scale rated 0–6, with one rating anchored for frequency, another rating the intensity of side effects encountered in the prior week that the study subject parents believed were due to the treatment, and the third asking the parents of study subjects to estimate the overall burden or degree of interference in day-to-day activities and function due to the side effects attributable specifically to the treatment [18
]. Frequency of side effects is rated as a percent time present: 0= no side effects; 1= present 10% of the time; 2=25% of the time; 3=50% of the time; 4=75%; 5=90%; and 6= present all of the time. Intensity of side effects ranges from 0= no side effects to 6= intolerable side-effects. Impairment due to side effects ranges from 0= no side effects to 6= unable to function at all due to side-effects. The PRISE lists a variety of possible side effects from which to choose and a scale to rate the specific side effect. The measure also has a place to list any side effects not previously listed.
Study subjects had lab testing collected at a Laboratory Corporation of American (LabCorp) draw station in the morning following an overnight fast. The lab was not made aware of the treatment status of the study subjects. The procedures for collection and analysis were defined by LabCorp standard protocols (CLIA-approved). The following blood tests were collected and evaluated on each study subject, including: whole blood white blood cell count (WBC), whole blood red blood cell count (RBC), whole blood platelet count, serum creatinine, serum blood urine nitrogen (BUN), serum alkaline phosphatase, serum aspartate aminotransferase (AST/SGOT), serum alanine aminotransferase (ALT/SGPT), serum glucose, and serum carnitine (total and free).
L-carnitine was supplied in a liquid preparation by the Wellness Pharmacy (Birmingham, AL, USA) using a specific formula containing: 100 mg L-Carntine/mL with the inactive ingredients of methylcellulose, stevioside (stevia), tangerine flavor, and preserved water (containing methylparaben and propylparaben). The placebos were identical in appearance and taste to the active preparation, containing a 1% methylcellulose suspension with the inactive ingredients of stevioside (stevia), tangerine flavor, and preserved water (containing methylparaben and propylparaben). The recommended childhood starting dose of 50 mg L-carnitine per Kg bodyweight per day (half the total dose administered in the morning and half the total dose administered in the evening) described in the package insert for CARNITOR® (Sigma-Tau Pharmaceuticals, Inc., Gaithersburg, MD, USA) was utilized in the present study with dosing calculated based on each participant’s initial weight. The dosing regimen of the liquid preparation was identical in both the L-carnitine and placebo groups, so that each study subject received a total of 0.5 mL per Kg of bodyweight per day (administered as 0.25 mL per Kg of bodyweight in the morning and 0.25 mL per Kg of bodyweight in the evening). Study subject-specific dosing instructions were placed on each liquid preparation provided to study subjects.
This was a randomized, double-blind, placebo-controlled study. The study was conducted between 2008 and 2010. The study subjects were recruited through community contacts. The study protocol called for 20 subjects to receive L-carnitine and 10 study subjects to receive placebo. summarizes the overall design of the present study. A total of 34 subjects were recruited for the present study. Four subjects withdrew prior to randomization into L-carnitine or placebo groups. A total of 30 subjects were randomly assigned to receive L-carnitine or placebo, and of these a total of 7 subjects (4 in the L-carnitine group and 3 in the placebo group) withdrew prior to successful completion of 3-months of therapy. Among the 7 subjects withdrawing from the study prior to successful completion of 3-months of therapy, 4 subjects withdrew because of adverse reactions (1 in the L-carnitine group and 3 in the placebo group), 2 subjects did not comply with the study protocol, and 1 was lost to follow-up with no known adverse reaction. Further, among the 4 subjects that withdrew because of adverse reactions, these patients were assessed using CARS and CGI scoring at the time of their withdrawal from the study.
Study subjects were shipped a 1 month supply of L-carnitine or placebo by Wellness Pharmacy, which automatically supplied subsequent refills on a monthly basis until each study subject received treatment for a total of 3-months. Laboratory, efficacy, and toleration evaluations were conducted on study subjects at baseline and following 3-months of therapy. In addition, study investigators monitored study subjects to ensure compliance and to monitor for potential adverse reactions.
Study subjects were seen for an initial screening where study investigators obtained information regarding demographics, formal diagnosis, age at diagnosis, age of apparent onset, information regarding delay or regression, any current medical issues, medications, bodyweight, and allergies on each study subject. A baseline CARS evaluation was performed by a single study investigator (JKK), an ATEC form was completed by the parents, hand muscle testing was performed by study investigators, and PRISE form was completed by the parents of study subjects. In addition, blood samples were collected on each study subject at a LabCorp draw station.
Following the initial screening and collection of labs, all study subjects started therapy within 30 days of baseline measurements. A study investigator (DAG), who did not perform any clinical measurements on study subjects, used a coin-flip to randomly assign study subjects to either the L-carnitine or placebo groups. Since there was a difference in sample size between the L-carnitine and placebo groups, the placebo group was filled with study subjects before the treatment group, so that the latter study subjects were all assigned to the L-carnitine group. Study investigators in contact with the study subjects and the parents of study subjects were not informed of the treatment status (L-carnitine/placebo) of each study participant until all study subjects had completed the trial, and hence the assignment (L-carnitine/placebo) strategy employed should not have revealed any information regarding the treatment status of any study participant to study investigators in contact with study subjects and the parents of study subjects. Subsequently, a study investigator (DAG) arranged with Wellness Pharmacy for shipment of appropriate study medication. For the duration of the trial any concomitant use of drugs/supplements were not changed as far as possible.
The primary lab efficacy measures were changes in total and free carnitine levels. Data were collected at baseline and at the end of the 3-months of treatment. In order to evaluate the clinical efficacy of treatment, CARS (determined by JKK) and ATEC (determined by the parents of the study subject) scores were generated at baseline and at the end of 3-months of treatment. In addition, an overall CGI score at the end of the 3-months of treatment was collected by a single study investigator (JKK). Finally, hand muscle strength (determined by a study investigator) was determined at baseline and at the end of the 3-months of treatment.
The primary lab tolerability measures were changes in WBC, RBC, whole blood platelet count, serum creatinine, serum BUN, serum alkaline phosphatase, serum AST/SGOT, serum alanine aminotransferase ALT/SGPT, and serum glucose. Data were collected at baseline and at the end of the 3-months of treatment.
A TAM form was completed by the parents of each study subject at the end of treatment. In addition, information was collected using a PRISE form completed by the parents of study subjects at baseline and at the end of 3-months of therapy, and a FISER/GRSEB form was completed by the parents of study subjects at the end of 3-months of therapy.
The statistical package contained in StatsDirect (Version 2.7.8) was utilized in the present study. All continuous variables were compared with the use of the parametric t-test statistic. Categorical variables were compared with the use of the Fisher’s exact test statistic. Outcomes measurements in the areas of efficacy and tolerability were evaluated as the relative change in tests results following 3-months of treatment in comparison to baseline measurements for each study subject. The null hypothesis was that there would be no difference in the data distributions of the relative change in test results following 3-months of treatment in comparison to baseline measurements between study subjects receiving L-carnitine in comparison to placebo. In addition, the simple linear-regression statistic was used to evaluate: for each study subject, regardless of his or her treatment status, the relationship between the change in serum free- carnitine levels following 3-months of treatment in comparison to his or her baseline measurements, and the changes in specific outcome measurements (hand muscle strength scores, cognitive scores measured using the ATEC, and CARS scores) 3-months of treatment in comparison to his or her baseline measurements. Further, 95% confidence intervals were determined for each linear regression line. The null hypothesis for each statistical test was that the slope of the line should be equal to zero. For all statistical tests performed in the present study, a two-tailed p-value ≤ 0.05 was considered to be statistically significant.