Clinical trial NCT05268744
Conditions: Autism Spectrum Disorder
Sponsor: University of California, San Francisco
Start date: 2022-11-14; Completion date: 2023-01-30
Enrollment: 16 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
An open-label examination of changes in metabolites with use of micronutrients in children with autism spectrum disorder (ASD). Investigators will also measure behavioral measures monthly at school with teachers and parents report.

Detailed description:
Children enrolled in Oak Hill School will enroll in this study and take micronutrient supplement once a day for 8 weeks period. Behavioral measures and metabolites taken from Dried Blood Spots (DBS) will be collected pre and post treatment. Behavioral measures will be taken again at 12 weeks, after 4 weeks of not dosing.

Arms: arm 1: In this open-label trial, all subjects will receive the daily dose of micronutrient, in the form of one lightning stick per day, which contains 2.9 grams of micronutrients per stick. It will be delivered in a powder form taken sublingually.
Interventions: intervention 1: Micronutrient
Intervention descriptions: intervention 1: Subject will take micronutrient supplement daily for 8 weeks

Protocol documents:
Prot_SAP_000.pdf: 
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Protocol: Micronutrient supplementation in children with autism spectrum disorder (ASD): A clinical 
trial examining mechanism of action 
NCT05268744 
Background: 
Prior studies have suggested that children with developmental disorders including autism spectrum 
disorder (ASD) have nutritional deficiencies and/or imbalances in vitamins and minerals, including low 
levels of vitamin D, lithium, zinc, and methionine and high levels of vitamin B6, vitamin C, and beta-
carotene, among others.1 These nutritional deficiencies may be responsible for impaired methylation, 
decreased glutathione levels, and increased oxidative stress seen in children with ASD.1  
The high prevalence of nutritional deficiencies in ASD may be due to dietary patterns in this population, 
and multiple studies indicate that children with ASD consume less varied diets with limited fruits and 
vegetables.2 Also, children with ASD have a high prevalence of gastrointestinal symptoms including 
diarrhea, constipation, and abdominal pain,3 which likely affects the absorption of nutrients and the 
composition of healthy bacteria in the intestinal tract (microbiome).  
There are likely functional consequences to these highly prevalent nutritional deficiencies. One analysis 
found that autism severity scores were correlated with levels of vitamins, minerals, and amino acids.1 A 
double-blind, randomized, placebo-controlled trial of a vitamin/mineral supplement in 141 children 
with autism found numerous improvements in nutritional biomarkers as well as a statistically significant 
improvement in parent ratings of child functioning (with specific questions on language, behavior, social 
function, sleep, gastrointestinal symptoms, and eye contact). The parent rating scale ranged from 1 to 7 
(lower scores better), and the children in the treatment group had a 0.34 greater improvement in this 
score (which was described as a modest benefit). There was no improvement in the Social 
Responsiveness Scale (SRS) or the Pervasive Developmental Disorder Behavior Inventory (PDD-BI).4 A 
subsequent study from the same group evaluated a more complex intervention that included a 
nutritional supplement as well as digestive enzymes, carnitine, essential fatty acids, Epsom-salt baths, 
and a healthy gluten-free, casein-free, soy free diet. The study found improvements in autism 
symptoms but was limited by the single-blind study design.5  
Micronutrient supplementation in other developmental disorders, including Attention 
Deficit/Hyperactivity Disorder (ADHD), have also shown clinical benefits. In a randomized controlled 
trial of 135 children (mean age 9.8) with ADHD, 54% of children treated with micronutrients were 
defined as 


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“responders” on a clinical global impression scale vs. only 18% of children in the placebo group. The 
treated children also had a small but significantly greater increase in height (6mm), but the parent 
ratings were not improved in the treatment group.6 
Several potential mechanisms of action of micronutrients supplementation on improved clinical 
outcomes have been proposed, including changes in gut microbiota, methylation capacity, and 
inflammation.7-9 Further clarification of the mechanism of action of micronutrient supplementation has 
the potential to identify specific pathways involved in clinical benefits and allow for more targeted 
treatment by screening children for nutritional deficiencies and modifying supplement formulations to 
optimize benefits. We previously demonstrated the value of using urinary metabolomics to identify 
physiological changes associated with clinical outcomes in a clinical trial of sulforaphane (an antioxidant 
supplement) in children with ASD.10 Fifteen children were enrolled and had a comprehensive urinary 
metabolomic analysis performed at baseline and after 8 weeks of treatment with sulforaphane. Five 
metabolic pathways – oxidative stress, amino acid/gut microbiome, neurotransmitters, hormones, and 
sphingomyelin metabolites were correlated with changes in clinical outcome measures. The 
sphingomyelin metabolite identification was novel and suggested a possible new line of research to 
determine whether antioxidant supplementation might alter this important component of neuronal 
development.10 
Since micronutrient deficiencies are common in children with ASD, and because there is scientific 
evidence to suggest that supplementation may produce important clinical benefits, we seek to more 
precisely examine the mechanism of action of micronutrient supplementation through a clinical trial 
using comprehensive, before-and-after metabolomic analyses. Since the prior study, the metabolomics 
field has advanced so that it is now possible to obtain metabolomic analyses using Dried Blood Spots 
(DBS), which are minimally invasive (requiring only a fingerstick and a drop of blood), and provide a 
much broader metabolomic analysis than urine. We therefore plan to use this new technology in the 
current study. The analyses may suggest specific metabolic changes that underlie clinical improvements 
in ASD, which may provide a greater understanding of the physiology of ASD and lead to improved, 
targeted treatment options.  
 
Methods:  


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Approvals: The clinical trial will be reviewed and approved by the UCSF Committee on Human Research 
prior to performing any study activities. The study protocol will be registered at clinicaltrials.gov. 
Participants: The UCSF investigative team has an ongoing relationship with a local, non-public school 
(Oak Hill School, San Anselmo, CA) that specializes in the education of children and young adults with 
autism and related neurodevelopmental disorders (ages 5-22, grades K-12). This unique academic-
school-parent partnership was created with the goal of improving overall care and communication 
between caregivers, clinical providers, and teachers. All children/families attending the school (n=50 
currently) will be invited to participate in the study through e-mail, informational flyers, and an evening 
informational session.  
Children and young adults will be eligible to participate if they are enrolled in the school, have a formal 
diagnosis of autism, report no use of micronutrient supplements within the last 3 months, are willing to 
hold other treatments constant for the 8-week study period, have no major medical problems other 
than ASD, are willing to provide urine samples, and parents were willing to complete on-line surveys at 
specified intervals. ASD is defined as being present if the child has a diagnosis from a medical 
professional trained to diagnose autism, or if the student is determined by school staff and the study 
psychiatrist to meet Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM–IV) 
criteria for ASD. Informed consent will be obtained from the parent/caregiver of all child study 
participants and directly from all adult study participants. Adults who are conserved will go through an 
informed consent process and provide assent, and their conservator will be asked to provide informed 
consent. 
Intervention: 
The planned study is open-label and all clinicians, parents, and teachers will be aware of the treatment 
initiation and duration of 8 weeks. All enrolled children will be provided a micronutrient 
supplementation (EM Power Plus). 
Objectives and outcomes: 
The primary goal of the study is to determine if any observed changes in symptoms of ASD are 
correlated with changes in metabolites that occur during treatment with micronutrients. We 
hypothesize that micronutrient treatment will lead to changes in physiological pathways and that those 
changes will correlate with clinical improvements. 


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The two primary outcome measure is the correlation of the changes in clinical symptoms with the 
change in specific metabolites. Two measures will be used to assess the change in core and associated 
features of autism: the Autism Behavior Inventory, Short Form (ABI-S) and the Social Responsiveness 
Scale (SRS). The ABI-S is an observer-reported outcome scale designed specifically to measure change 
and severity of ASD symptoms and has been shown to have content validity when completed by 
caregivers.11  The SRS is one of the most commonly used outcome measures in clinical trials of ASD and 
focuses on social responsiveness and interaction.12 Parents/caregivers will be asked to complete both 
measures at baseline, 4 weeks, and 8 weeks using an on-line and secure platform. 
Dried blood spots (DBS) will be collected on the day of study initiation and on the final day of the study 
after the 8-week treatment period. We will be using “batch enrollment” where the parents and 
caregivers of all potentially eligible participants will be screened for eligibility and asked to go through 
an informed consent process. Once the final “set” of eligible student participants is identified, they will 
all be given the study medication and have the DBS sample collected on the same day. DBS is collected 
using a simple, sterile, finger-prick device and technique, which generates a drop of blood. The 
participant will be assisted to place his/her finger on a special DBS “card” which will collect the drop of 
blood. DBS cards will be individually packaged and stored immediately at -80 °C. During the screening 
visit, children will have a brief physical examination including height and weight to guide the proper 
dosing. Micronutrient treatment will be provided at the screening visit along with dosing instructions, 
but participants will not begin the treatment until the study initiation day, when the DBS samples are 
collected.  
Safety assessments: 
Parents/caregivers and teachers will be advised to report any concerns about a new medical problem 
immediately to the study investigators, who will be available at all times to receive reports of possible 
adverse effects. At the 4 week and 8-week on-line questionnaires, parents/caregivers will be asked to 
report any new medical problems or concerns for possible side effects.  
Metabolomic analyses:  
All DBS samples will be sent on dry ice in one batch to Metabolon (Morrisville, NC). Metabolomics 
analyses will be conducted at Metabolon as previously described 13.  Briefly, samples will be subjected to 
methanol extraction, then split into five aliquots for analysis by ultrahigh performance/mass 
spectrometry in the positive (two methods), negative or polar ion modes.  Metabolites will be identified 


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by automated comparison of ion features to a reference library of chemical standards followed by visual 
inspection for quality control 14. For statistical analyses and data display, any missing values will be 
assumed to be below the limits of detection; these values will be imputed with the compound minimum 
(minimum value imputation).  Data will then be normalized by measured osmolality, which is necessary 
to reduce the variability 15. A metabolic pathway for a given metabolite will be assigned based on prior 
designations in the literature combined with experience from prior datasets at Metabolon.  
 
Statistical methods:  
Summary statistics will be used to describe the variables.  Change in both the clinical variables and the 
metabolites will be computed as post-test minus pre-test.  Pearson’s correlation coefficient will be 
estimated and tested to index the association between the change in each of the two clinical scales and 
the change in each metabolite. Given the early-stage nature of the research, we will not adjust for 
multiple comparisons because we believe it was more important to risk a Type II error than to miss a 
potentially important signal by being overly conservative, as has been suggested by prior authors 16.   
We define, a priori, that a correlation cutoff of an absolute value of > 0.6 is of potential clinical 
relevance, because, given our planned sample size of 25, we will have approximately 80% power to 
detect a correlation of that size and such a correlation accounts for roughly one-third of the variance. 
Others have suggested that correlations with an absolute value of > 0.6 indicate a moderate or higher 
correlation 17 (and are hence of greatest interest in pointing to a mechanism of action). 
We also plan to examine the number of participants who have a “clinical response,” defined a priori as 
an improvement of 4 or more points in the ABI-S. We will compare the pre-post changes in the ABI-S 
and SRS in the responder and non-responder groups using the student’s t-test.  
 
 


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Planned Data Tables: 
 
Table 1: Characteristics of Enrolled Participants 
 Category 
Characteristic 
Subjects 
N=15 
  
  
% 
N 
Gender 
Male 
 
 
 
Female 
 
 
Ethnicity 
White 
 
 
Asian/Pacific Islander 
No response 
Age 
7-10 
11-14 
15-21 
Mean Age 
Primary diagnosis 
Autism Spectrum Disorder 
Comorbidities 
Intellectual Disability 
Language Disorder 
ADHD 
Pica 
 
Global Development Delay 
 
 
Learning Disability 
 
Other 
 
 
Current meds 
Sertraline 
Lurasidone 
Risperidone 
Birth Control 
Zonisamide 
 
Other 
 
 
 


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Table 2: Metabolite correlations 
 
Primary Outcome Measure 
 
Metabolite 
ABI-S 
Corr.     p-value 
SRS 
Corr.     p-value 
Metabolic Pathway 
Amino acids (endogenous) 
 
 
 
  
 
 
Benzene metabolism 
  
  
Cholesterol metabolism 
  
  
Fatty acids 
  
  
Monoterpene phenol 
  
  
Neurotransmitters 
  
  
Oxidative stress 
  
  
Polyol 
 
  
  
 
 
 
Sphingomyelin 
  
  
Sugars 
  
  
TCA cycle 
  
  
Other 
 
  
 
 
 
 
 
 


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Table 3. Change in outcome scores over the 12-week study period 
Outcome Measure 
Adjusted Mean Scores (95% CI) 
Change from Baseline (95% CI) 
 
Baseline 
1 month 
3 months 
1 month 
p 
3 
months 
p 
Autism Behavior 
Inventory 
Total Score 
 
 
 
 
 
 
 
Social 
Communication 
 
 
 
 
 
 
 
Restrictive and 
Repetitive Behaviors 
 
 
 
 
 
 
 
Mental Health 
 
 
 
 
 
 
 
Self-regulation 
 
 
 
 
 
 
 
Challenging 
Behaviors 
 
 
 
 
 
 
 
Social Responsiveness 
Scale 
Total Score 
 
 
 
 
 
 
 
Awareness 
 
 
 
 
 
 
 
Cognition 
 
 
 
 
 
 
 
Communication 
 
 
 
 
 
 
 
Mannerisms 
 
 
 
 
 
 
 
Motivation 
 
 
 
 
 
 
 
Mean change scores will be adjusted for age and gender; *indicates statistically significant change from 
baseline 
 
 
 
 
 
 


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