You are on page 1of 13

Sulforaphane treatment of autism spectrum

disorder (ASD)
Kanwaljit Singha,b, Susan L. Connorsa, Eric A. Macklinc, Kirby D. Smithd, Jed W. Faheye, Paul Talalaye,1,
and Andrew W. Zimmermana,b,1
a
Lurie Center for Autism, Department of Pediatrics, Massachusetts General Hospital for Children, Harvard Medical School, Lexington, MA 02421; bDepartment
of Pediatrics (Neurology), University of Massachusetts Medical School, Worcester, MA 01655; cDepartment of Medicine, Massachusetts General Hospital
Biostatistics Center and Harvard Medical School, Boston, MA 02114; and dMcKusick–Nathans Institute for Genetic Medicine and eDepartment of
Pharmacology and Molecular Sciences, Lewis B. and Dorothy Cullman Chemoprotection Center, The Johns Hopkins University School of Medicine, Baltimore,
MD 21205

Contributed by Paul Talalay, September 4, 2014 (sent for review August 12, 2014; reviewed by Bryan H. King, Robert K. Naviaux, and Cecilia Giulivi)

Autism spectrum disorder (ASD), characterized by both impaired by sulforaphane, thus reducing the risks of developing malig-
communication and social interaction, and by stereotypic behav- nancies and other chronic diseases (5–10). Sulforaphane is now
ior, affects about 1 in 68, predominantly males. The medico- in widespread clinical evaluation (10).
economic burdens of ASD are enormous, and no recognized treat- The decision to test sulforaphane to treat ASD was based on
ment targets the core features of ASD. In a placebo-controlled, four premises. First, extensive evidence shows that sulforaphane
double-blind, randomized trial, young men (aged 13–27) with counteracts many of the same biochemical and molecular ab-
moderate to severe ASD received the phytochemical sulforaphane normalities associated with ASD, including oxidative stress and
(n = 29)—derived from broccoli sprout extracts—or indistinguish- reduced antioxidant capacity, defects in glutathione synthesis,
able placebo (n = 15). The effects on behavior of daily oral doses mitochondrial dysfunction and low oxidative phosphorylation,
of sulforaphane (50–150 μmol) for 18 wk, followed by 4 wk without
increased lipid peroxidation, and neuroinflammation (11–16).
treatment, were quantified by three widely accepted behavioral
Although it is unclear whether these anomalies are etiological or
measures completed by parents/caregivers and physicians: the Ab-
secondary manifestations, their correction often improves ASD
errant Behavior Checklist (ABC), Social Responsiveness Scale (SRS),
and Clinical Global Impression Improvement Scale (CGI-I). Initial
behavior (17).
scores for ABC and SRS were closely matched for participants
Second, a variety of small molecules including sulforaphane can
assigned to placebo and sulforaphane. After 18 wk, participants ameliorate a number of unrelated genetic disorders by activating
receiving placebo experienced minimal change (<3.3%), whereas the “stress proteome,” which regulates many of the aforemen-
those receiving sulforaphane showed substantial declines (improve- tioned damaging processes. Sulforaphane, as well as hydroxyurea,
ment of behavior): 34% for ABC (P < 0.001, comparing treatments)
and 17% for SRS scores (P = 0.017). On CGI-I, a significantly greater Significance
number of participants receiving sulforaphane had improvement
in social interaction, abnormal behavior, and verbal communi- Autism spectrum disorder (ASD), encompassing impaired
cation (P = 0.015–0.007). Upon discontinuation of sulforaphane, communication and social interaction, and repetitive stereo-
total scores on all scales rose toward pretreatment levels. Dietary typic behavior and language, affects 1–2% of predominantly
sulforaphane, of recognized low toxicity, was selected for its male individuals and is an enormous medical and economic
capacity to reverse abnormalities that have been associated with problem for which there is no documented, mechanism-based
ASD, including oxidative stress and lower antioxidant capacity, treatment. In a placebo-controlled, randomized, double-blind
depressed glutathione synthesis, reduced mitochondrial function clinical trial, daily oral administration for 18 wk of the phyto-
and oxidative phosphorylation, increased lipid peroxidation, and chemical sulforaphane (derived from broccoli sprouts) to 29
neuroinflammmation. young men with ASD substantially (and reversibly) improved
behavior compared with 15 placebo recipients. Behavior was

A utism spectrum disorder (ASD) includes neurodevelopmental


abnormalities characterized by impaired ability to communi-
cate and interact socially and by restricted and repetitive patterns
quantified by both parents/caregivers and physicians by three
widely accepted measures. Sulforaphane, which showed neg-
ligible toxicity, was selected because it upregulates genes that
of behavior, interests, and activities (1). The prevalence of ASD in protect aerobic cells against oxidative stress, inflammation,
the United States is about 1 in 68 among children aged 8 y, with and DNA-damage, all of which are prominent and possibly
marked male (4.5:1) preponderance (2). No validated pharmaco- mechanistic characteristics of ASD.
logical treatments for the core symptoms of ASD are available. We
report here that in a placebo-controlled, double-blind, randomized Author contributions: K.D.S., P.T., and A.W.Z. designed research; K.S., S.L.C., and A.W.Z.
performed research; J.W.F. and P.T. contributed new reagents/analytic tools; K.S., E.A.M.,
clinical trial, daily treatment with sulforaphane for 4–18 wk resulted J.W.F., P.T., and A.W.Z. analyzed data; K.S., K.D.S., J.W.F., P.T., and A.W.Z. wrote the
in significant improvements in aberrant behavior and social paper; and J.W.F. and P.T. supplied sulforaphane-rich broccoli sprout extract.
impairment in a majority of young males diagnosed with moderate Reviewers: B.H.K., Seattle Children’s Autism Center; R.K.N., University of California,
to severe autism, and that this improvement regressed upon ces- San Diego; and C.G., University of Southern California.
sation of treatment. Physician and parent/caregiver impressions of Conflict of interest statement: U.S. patent applications have been filed by The Johns
clinical improvement were evaluated by behavioral outcome Hopkins University (inventors K.D.S., P.T., and A.W.Z.). P.T. and A.W.Z. have divested
measures. themselves from all potential financial benefits. The sulforaphane-rich broccoli sprout
extract is not a commercial product. Broccoli sprouts and seeds rich in glucosinolates
Sulforaphane is an isothiocyanate derived from broccoli. Its have been licensed by Johns Hopkins to Brassica Protection Products LLC (A. Talalay,
therapeutic potential is based on its potent activity in transcrip- son of P.T., is chief executive officer).
tionally up-regulating genes that control mechanisms whereby Freely available online through the PNAS open access option.
aerobic cells protect themselves against oxidative stress, in- 1
To whom correspondence may be addressed. Email: ptalalay@jhmi.edu or Andrew.
flammation, DNA-damaging electrophiles, and radiation (3, 4). Zimmerman@umassmemorial.org.
Under basal conditions, these protective systems do not operate This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
at maximal capacity but can be induced to higher activity levels 1073/pnas.1416940111/-/DCSupplemental.

15550–15555 | PNAS | October 28, 2014 | vol. 111 | no. 43 www.pnas.org/cgi/doi/10.1073/pnas.1416940111


phenylbutyrate, and trichostatin A, have been shown in vitro to abnormalities in physical examination, blood chemistries, hema-
have therapeutic potential to reestablish cellular homeostasis in tology, and urinalysis (Table S1).
a number of unrelated genetic disorders (18).
Third, sulforaphane is a dietary phytochemical, derived from its Analysis of Outcome Measures. The total and the changes in total
precursor glucosinolate glucoraphanin, that is widely consumed in ABC and SRS behavioral scores of the 26 sulforaphane-treated
cruciferous plant-rich diets, and qualifies for consideration as and 14 placebo recipients from enrollment to the 18-wk end of
a food, a dietary supplement, or a drug, depending on its treatment and after a 4-wk recovery period are shown in Figs. 2–4
intended use. Sulforaphane is therefore justifiably considered and Tables 1 and 2. Treatment group mean ABC scores differed
to be of low toxicity, and its administration to humans is well significantly at 4, 10, and 18 wk (Fig. 2 B and E for ABC and SRS,
tolerated (10, 19, 20). respectively). At 18 wk there was a 34% reduction in ABC and
Fourth, widespread anecdotal reports have suggested that fe- a 17% reduction in SRS scores, and these trended toward non-
ver can dramatically but temporarily ameliorate the disturbed significant differences upon cessation of treatment (Fig. 2 B and
behavior of many autistic patients (21). Notably, the degree of E and Tables 1 and 2).
improvement (mostly in stereotypic behavior and inappropriate Significantly greater improvement was observed among par-
speech) was unrelated to the severity of fever or of autism (21). ticipants randomized to sulforaphane at 4, 10, and 18 wk for irri-
This study explicitly suggested that elucidation of the fever re- tability, lethargy, stereotypy, and hyperactivity subscales of the
sponse might provide insight into the mechanisms of ASD and ABC, and in awareness, communication, motivation, and man-
point to new therapeutic approaches (21, 22). Fever up-regulates nerism subscales of SRS (Fig. 3 and Tables 1 and 2). After stopping
heat-shock proteins and related mechanisms central to multiple sulforaphane treatment, both ABC and SRS subscores tended to
cellular processes in the CNS, including synaptic transmission revert toward baseline.
(23, 24), and may improve long-range cerebral cortical connec- On subscale analysis of CGI-I scale scores at 18 wk (Tables 1
tivity that is depressed in ASD (25). Sulforaphane also up- and 2), 46% (12 of 26), 54% (14 of 26), and 42% (11 of 26) of
regulates expression of the heat-shock response (26). sulforaphane recipients were much or very-much improved on
We hypothesized that daily treatment with sulforaphane at social interaction, aberrant behavior, and verbal communication,
levels achieved by diet might reduce the severity of socially im- respectively, compared with 0% (0 of 11; P = 0.007), 9% (1 of 11;
paired behavior in ASD. Behavior was quantified directly by three P = 0.014), and 0% (0 of 11; P = 0.015), respectively, for
widely validated behavioral outcome measures at the periods placebo recipients.
before, during, and after intervention (Fig. 1). Parents/caregivers Individual changes in total ABC and SRS scores from basal
completed the Aberrant Behavior Checklist (ABC) (27) and the levels to 18 wk are shown in Fig. 4. A positive response was de-
Social Responsiveness Scale (SRS) (28). Study physicians com- fined post hoc as a 30% decrease from baseline in total ABC and
pleted the Clinical Global Impression Severity (CGI-S) and the SRS scores. Thirty-five percent (9 of 26) of participants on sul-
Clinical Global Impression Improvement (CGI-I) scales (29, 30). foraphane had a positive response on SRS compared with 0% (0
of 11) on placebo (Fisher’s exact test P = 0.036), and 60% (15 of
Results 25) of participants receiving sulforaphane had a positive response
Participant Characteristics. More than 90% of all scheduled tests on ABC compared with 20% (2 of 10) on placebo (P = 0.059).
were completed on the 40 participants who received placebo or Our clinical impressions during the study, although blind to
sulforaphane treatment and returned for the first return visit group assignment, were that 13 of the 40 participants improved
(week 4). Twenty-two participants (6 placebo, 16 sulforaphane) noticeably with respect to sociability and behavior, usually ob-
were also tested at 22 wk, 4 wk, after treatment ended (Table 1 servable by 4 wk; all were receiving sulforaphane. In queries to
and Fig. S1). Four participants (one placebo, three sulforaphane) families and caregivers, before unblinding, 17 of 26 whose sons
were lost to follow-up before their first on-treatment visit. had taken sulforaphane reported gradual changes within the first
Participants, all male, were 13–27 y old at enrollment (median: month of treatment and correctly surmised their group assign-
17 y). A history of behavioral improvements with fever was given ment, whereas the remaining 9 on sulforaphane—and all but 1 of 14
by a large majority (32 of 40; 80%) of participants. Participants who received placebo—were not improved, and believed that
in sulforaphane and placebo groups were well matched, and did their sons had not received sulforaphane. Positive responses to
not differ at baseline with respect to various demographic, be- sulforaphane were spontaneously reported by parents and care-

MEDICAL SCIENCES
havioral and clinical features, behavioral outcome score measures, takers, who commented (before disclosure of treatment cate-
gory) on improved social responsiveness, behavioral compliance,
and calmness in the subjects with ASD who were taking the
active compound.

Safety and Adverse Events. Sulforaphane treatment effectively


improved core aberrant behaviors of ASD, and was safe and
well-tolerated (Table S2). Notably, none of the laboratory
results were outside normal ranges at any time point (Dataset
S1). Unexpectedly, the sulforaphane group gained significantly
more weight over the 18-wk period, compared with placebo
(4.31 vs. 0.31 lb, P = 0.056). Pulse rate was lower in the sul-
foraphane group both at baseline and during the study. Thirty-
six adverse events were noted during the trial. Vomiting, increased
aggressions, abdominal pain, increased flatulence, irritability, con-
stipation, diarrhea, fever, headache, and exacerbation of seasonal
allergies were reported in 12–19% of participants on sulforaphane;
their incidence was the same in the placebo groups (P > 0.10).
Two participants had single unprovoked seizures: one after
3 wk on sulforaphane, with an undisclosed history of recent
seizures; the other 3 wk after discontinuing treatment and a past
Fig. 1. Schedule for study of the effects of sulforaphane in ASD. (more than 1 y) history of seizures well-controlled with antiepileptic

Singh et al. PNAS | October 28, 2014 | vol. 111 | no. 43 | 15551
Table 1. Effect of sulforaphane treatment on Aberrant Behavior Checklist (ABC) and Social Responsiveness Scale
(SRS) scores
Total and changes in mean total scores

Time of observations (wk)

Scale and treatment 0 4 10 18 22

ABC
Placebo
Baseline 60.14 59.77 58.85 58.10 57.67
Intervention point 60.14 60.54 62.15 56.10 55.83
Change* 0 0.77 ± 1.84 3.31 ± 3.50 −2.00 ± 4.59 −1.83 ± 6.60
n 14 13 13 10 6
Sulforaphane
Baseline 62.77 62.77 62.34 63.88 69.16
Intervention point 62.77 50.08 42.73 42.44 58.44
Change* 0 −12.69 ± 4.17 −19.61 ± 5.95 −21.44 ± 4.34 −10.72 ± 5.07
n 26 26 22 25 16
P value (between treatments)† – 0.035 0.002 <0.001 0.33
SRS
Placebo
Baseline 120.21 120.21 118.85 119.55 122.00
Intervention point 120.21 112.43 117.46 117.55 115.33
Change* 0 −7.79 ± 3.09 −1.38 ± 3.72 −2.00 ± 3.46 −6.67 ± 3.82
n 14 14 13 11 6
Sulforaphane
Baseline 120.15 120.88 118.26 120.96 116.91
Intervention point 120.15 106.12 103.78 100.56 109.88
Change* 0 −14.76 ± 3.79 −14.48 ± 5.72 −20.40 ± 4.54 −7.03 ± 4.20
n 26 25 23 25 16
P value (between treatments)† – 0.29 0.080 0.017 0.87

ABC and SRS total scores of participants who completed at least one postintervention measurement (n = 40). The ABC and SRS scores
and changes thereof from baseline are the raw, unadjusted values and the P values are from the linear mixed model adjusting for
repeated measures.
*Individuals’ scores at 4, 10, 18, or 22 wk were subtracted from the same individual’s scores at time 0 (“Baseline”); differences were
averaged, and are presented as means ± SEM. Because the number of individuals for whom scores were obtained (n) at each time
period varied, so did the baseline score used to calculate each change.

P values as determined from mixed-effects general linear model.

drugs. Although patients with autism are predisposed to seizures contrast, this study was, to our knowledge, one of the few designed
(31, 32), we cannot rule out the possibility of seizures as an adverse to target core clinical features as well as the fundamental bio-
effect of sulforaphane in ASD. chemical abnormalities of ASD (oxidative stress and antioxidant
deficiency, increased susceptibility to electrophile toxicity, and
Discussion inflammation) by the administration of sulforaphane.
The behavioral outcome measures (ABC, SRS, CGI-I) and clini- Our suggestion that participants with ASD whose behavior
cal observations by study physicians and many parents/caregivers, improved during fever would also respond to sulforaphane could
all before unmasking, indicated that many of the participants who not be confirmed because of the unusually high prevalence of
were treated with sulforaphane in this study had statistically sig- fever responders (80%) in our cohort compared with most ASD
nificant and clinically meaningful improvements during treatment populations (35%) (33). Unlike the rapid onset of changes in
with sulforaphane. The substantial improvements of individual behavior during fever in ASD, responses to sulforaphane in this
ASD patients’ trajectories were conspicuous and suggest that study appeared over several weeks. This finding suggests that
further investigation of sulforaphane in ASD is promising. sulforaphane may cause increases in gene transcription in multiple
Although we observed consistent and large improvements in underperforming cell-signaling pathways (34, 35). Sulforaphane
behavior in the majority of sulforaphane-treated ASD, this was may be only one of several small molecules that will ameliorate
a single-site, limited dose-range study of only 44 male, pre- deficiencies that lead to abnormal functioning in the whole or-
dominantly Caucasians, aged 13–27 y, 4 of whom dropped out of ganism. Further studies of sulforaphane’s effects at the cellular
the study before their first follow-up visit. Although we did not test level, if confirmed, could guide discovery of new drugs with
specifically for adaptive or cognitive skills at baseline, our cohort similar underlying mechanisms of action in ASD.
included subjects with moderate and severe ASD with substantial Activation of the cellular stress response, in addition to its
variability in total baseline SRS and ABC scores (Fig. 4 and Tables therapeutic potential, is known to protect cells from environ-
1 and 2). Because of considerable heterogeneity in the etiology, mental toxins. Recently, we demonstrated the efficacy of sulfor-
pathogenesis, and symptomatology of ASD, generalization of our aphane as an environmental detoxicant (36). Together with sul-
findings requires confirmation. foraphane’s capacity to activate the Keap1–Nrf2 cytoprotective
Nevertheless, this study may shed light on the basic patho- signaling pathway (10), it may therefore protect against both en-
physiology of at least a subset of ASD. Most clinical studies and vironmental and endogenous risk factors that affect brain de-
medications aim to restrain ASD’s troublesome symptoms. In velopment in ASD (37). Given its favorable safety profile, future

15552 | www.pnas.org/cgi/doi/10.1073/pnas.1416940111 Singh et al.


Fig. 2. Changes in total ABC and SRS scores. Forty male ASD participants who were treated daily with either placebo (initially n = 14) or sulforaphane
(initially n = 26) for 4, 10, and 18 wk, followed by a terminal 4-wk untreated period (22 wk). Panels A (ABC) and D (SRS) show all observations. Means of
changes in raw, unadjusted total scores (±SEM) at 4, 10, 18, and 22 wk are shown in B for ABC and E for SRS. Reductions in ABC score upon sulforaphane
treatment were −20.2% (P = 0.035), −31.5% (P = 0.002), and −33.6% (P < 0.001), at 4, 10, and 18 wk, respectively. The corresponding changes in SRS were −12.2%
(P = 0.29), −12.2% (P = 0.080), and −16.8% (P = 0.017). Panels C (ABC) and F (SRS) show the changes in total scores at all timepoints for placebo- and sulforaphane-
treated participants. All changes were calculated from the initial values for each individual participant at time 0 (the means of the two values obtained at
screening and at enrollment).

studies should address sulforaphane’s potential benefits for the Materials and Methods
prenatal prevention of ASD as well as for the early treatment of Study Protocol. This study was conducted at the Lurie Center for Autism of the
young children with this disorder. Massachusetts General Hospital (MGH) for Children with approval of the

MEDICAL SCIENCES
Table 2. Clinical Global Impression-Improvement (CGI-I) scores at 18 wk for the 37 subjects for
whom scores were available
Number of subjects’ scored as either “much improved” or
“very much improved” after 18 wk/total number of
subjects (% of total number evaluated)

Subscore Placebo Sulforaphane P for-difference*

Overall level of autism 0/11 (0%) 0/26 (0%) –


Social interaction 0/11 (0%) 12/26 (46.2%) 0.007
Aberrant/abnormal behavior 1/11 (9.1%) 14/26 (53.8%) 0.014
Repetitive and stereotypical behavior 0/11 (0%) 6/26 (23.1%) 0.15
Verbal communication 0/11 (0%) 11/26 (42.3%) 0.015
Nonverbal communication 1/11 (9.1%) 5/26 (19.2%) 0.65
Hyperactivity and inattention 0/11 (0%) 3/26 (11.5%) 0.54
Anxiety 0/11 (0%) 2/26 (7.7%) >0.99
Sensory sensitivities 0/11 (0%) 6/26 (23.1%) 0.15
Restricted and narrow interests 0/11 (0%) 0/26 (0%) –

*By Fisher exact test.

Singh et al. PNAS | October 28, 2014 | vol. 111 | no. 43 | 15553
thyroid functions. Participants continued their regular medications, if any,
during the study.
Participants were assigned by the MGH Research Pharmacy to receive
either placebo or sulforaphane according to computer-generated randomly
permuted blocks of three assignments, with sulforaphane and placebo
treatments allocated in a 2:1 ratio in two strata defined by parent-reported
history of improvement in behavior during febrile illness. Physicians and study
staff were blind to group assignment. Forty-four subjects were selected to
provide at least 80% power to test the primary hypothesis for the SRS using
a two-tailed two-sample t test with α = 0.05 and assuming that the true
difference in average change in SRS was 15 units with a SD of 16 units. This is
roughly twice the average magnitude of natural change observed over 1 y
among male children and adolescents with ASD (38).
The study comprised seven visits: screening, randomization, and start of
treatments, at 24 h, and at 4, 10, and 18 wk after the first dose. Treatment was
discontinued after the 18-wk visit, and participants returned at 22 wk.
Medical history, physical examination including vital signs, adverse event
reporting, and SRS, ABC, and CGI-I were performed (Fig. 1). At the 4-, 18-, and
22-wk visits, hematology, chemistry, and urinalysis were also obtained.
All families were contacted after the final participant completed follow-up
and asked for their impressions of the study and their child’s progress
while under treatment. Families were then informed whether he received
sulforaphane or placebo.
Fig. 3. Changes in ABC subscores for irritability, lethargy, stereotypy, and
hyperactivity. After 4, 10, and 18 wk of treatment with sulforaphane or
Administration of Medication and Protocol Schedule. Capsules of sulforaphane-
placebo, and a 4-wk untreated recovery period (22 wk). Raw, unadjusted
mean values of changes (±SEM) for sulforaphane- and placebo-treated rich broccoli sprout extracts were maintained at −20 °C, and checked period-
participants are shown. Changes were significant at the 95% confidence ically microbiologically and for sulforaphane titer (SI Materials and Methods)
level (*) for both irritability and lethargy at 10 and 18 wk of treatment. (8). Indistinguishable placebo capsules contained microcrystalline cellulose.
Sulforaphane or placebo was administered daily for 18 wk. The participants
were dosed according to body weight: 50 μmol (one capsule) of sulforaphane
MGH and Johns Hopkins University Institutional Review Boards, and was for <100 lb, 100 μmol (two capsules) for 101–199 lb, and 150 μmol (three
capsules) for >200 lb. Placebo recipients received equivalent numbers of cap-
registered at ClinicalTrials.gov (NCT 01474993 under Food and Drug Ad-
sules according to their weight. Capsules were dispensed to participants in
ministration IND 113542). All participants who were able, and parents or
sealed bottles by the MGH Research Pharmacy, with instructions to keep them
caregivers, gave written informed consent. All participants met criteria for
in a household freezer.
autistic disorder (1). Forty-four male ASD patients were enrolled from
February 2011 to July 2013. The Autism Diagnostic Observation Schedule, per-
Behavioral Outcome Measures. The ABC is a parent- or caregiver-reported
formed by a trained psychologist/tester (in 43) or DSM-4 (1) checklist of 58-item questionnaire designed to assess medication effects; each item is
symptoms performed by a trained physician (two participants), were used to scored on a scale of increasing severity from 0 to 3 (27). ABC also assesses several
confirm the diagnosis of autism at the screening visit. All participants were subdomains (irritability, lethargy, stereotypy, and hyperactivity).
moderately to severely autistic on the CGI-S, with varied cognitive capacity The SRS is a parent- or caregiver-reported 65-point social communication
(Table S1). questionnaire that covers five subscales (awareness, cognition, communica-
Eligibility criteria included male sex, age 13–30, no intercurrent chronic tion, motivation, and autistic mannerisms) (28). Each SRS item is rated on a
illness, no history of active seizures within 1 y, and normal liver, renal, and scale of 1–4; the total score was our primary efficacy endpoint.

Fig. 4. Total scores for (A) ABC and (B) SRS of individual placebo- and sulforaphane-treated participants at baseline and after 18 wk. At 18 wk, total ABC
scores were available for 35 (10 placebo and 25 sulforaphane) and total SRS scores for 37 (11 placebo and 26 sulforaphane). Only the differences for sul-
foraphane treatment were significant at 18 wk, thus a change in score of from 62.4 to 45.0 on the ABC scale (A) was significant (P < 0.001), and a change in
score of from 121.5 to 105.2 on the SRS scale (B) was significant (P < 0.001). Means for the subjects shown, at 1 and 18 wk respectively, for placebo treatment,
were 62.4 and 62.6 on the ABC scale, and 121.5 and 117.5 on the SRS scale.

15554 | www.pnas.org/cgi/doi/10.1073/pnas.1416940111 Singh et al.


The Ohio Autism Clinical Global Impression Severity Scale (CGI-S, also des- visit and treatment group and random participant-specific intercepts and slopes
ignated OACIS-S, and only measured at screening) (29, 30) is a clinician-rated with unstructured covariance. The absence of a main effect for treatment (i.e.,
assessment of the severity of autistic behavior (in increasing order of severity a “shared baseline”) properly reflects the true state of the population sampled
from 1 to 7) and includes the following subdomains: global autism severity, before randomization and has the advantage of adjusting for any chance dif-
social interaction, aberrant behavior, repetitive or ritualistic behaviors, verbal
ferences at baseline in a manner similar to ANCOVA (39). Linear contrasts of
and nonverbal communication, hyperactivity/inattention, anxiety, sensory
least-square means were used to estimate changes from baseline between
sensitivities, and restricted/narrow interests. The Ohio Autism Clinical Global
Impressions Improvement Scale (CGI-I or OACIS-I) (29, 30) is a clinician-rated treatment and control groups at each follow-up visit. Given its assumptions,
assessment of how much the patient’s behavior has changed during an the mixed model yields estimates that are unbiased as long as loss to follow-
intervention. up, and missing test scores are predictable from observed scores under
assumptions of the model. An intention-to-treat analysis that included all
Statistical Evaluation. Forty-four subjects were originally enrolled and ran- 44 participants led to similar conclusions (SI Materials and Methods).
domized to sulforaphane treatment (n = 29) or placebo (n = 15); four Statistical analyses were performed with SAS v. 9.3 software (SAS Institute),
subjects discontinued participation in the study before the first (4-wk) and Stata v.11.2 (Statacorp).
return visit. Behavior scores for the remaining 40 participants, who com-
pleted at least part of the outcome measure evaluations (14 placebo and ACKNOWLEDGMENTS. We thank the participants and their families who
26 sulforaphane), are described in our primary results and shown in Figs. 2–4 were consistently interested and gave generously of their time; Scott Zeger
and Tables 1 and 2. To compensate for incidental changes in ABC/SRS scores for discussions on biostatistics; Jessica Helt and Karmen Koesterer for patient
resulting from normal fluctuation, we obtained these scores at both screening testing and Luisa Masclans for data collection; Ann Neumeyer, who chaired
and randomization visits, and used their averages to compare with subsequent the Data Safety Monitoring Board and was consulted regarding safety and
ABC/SRS scores. Our primary analysis used the differences between scores of side effects; Jennifer Mullett for assisting with study procedures; and
individuals at 4, 10, 18, and 22 wk from their respective average pretreatment Christine Ferrone and Lisa Nowinski for advising us on regulatory matters
and outcome measures. The quality of data collection, retrieval, and analysis
values. The test of our hypothesis was the difference between the sulfor-
were certified by Quality Associates Incorporated. The study was supported
aphane and placebo treatment groups in the change in ABC and SRS scores by gifts from the Nancy Lurie Marks Family Foundation, the Hussman
from baseline to 18 wk, and their reversion to baseline at 22 wk. Foundation, the Lewis B. and Dorothy Cullman Foundation, the Agnes Gund
Each outcome was modeled in a shared-baseline mixed-effects general Foundation, the N of One Foundation, and the Brassica Foundation for
linear model with fixed effects for visit and the interaction of postrandomization Chemoprotection Research.

1. American Psychiatric Association (2000) Diagnostic and Statistical Manual of Mental 20. Fahey JW, Kensler TW (2013) Health span extension through green chemoprevention.
Disorders, 4th edition (DSM-IV) (American Psychiatric Association, Arlington, VA). Virtual Mentor 15(4):311–318.
2. Developmental Disabilities Monitoring Network Surveillance Year 2010 Principal 21. Curran LK, et al. (2007) Behaviors associated with fever in children with autism
Investigators; Centers for Disease Control and Prevention (CDC) (2014) Prevalence spectrum disorders. Pediatrics 120(6):e1386–e1392.
of autism spectrum disorder among children aged 8 years—Autism and de- 22. Mehler MF, Purpura DP (2009) Autism, fever, epigenetics and the locus coeruleus.
velopmental disabilities monitoring network, 11 sites, United States, 2010. MMWR Brain Res Brain Res Rev 59(2):388–392.
Surveill Summ 63(2):1–21. 23. Stetler RA, et al. (2010) Heat shock proteins: Cellular and molecular mechanisms in the
3. Zhang Y, Kensler TW, Cho CG, Posner GH, Talalay P (1994) Anticarcinogenic activities central nervous system. Prog Neurobiol 92(2):184–211.
of sulforaphane and structurally related synthetic norbornyl isothiocyanates. Proc 24. Hollander E (2013) Translational Experimental Therapeutics of Inflammation and
Natl Acad Sci USA 91(8):3147–3150. Fever in Autism Spectrum Disorder: Hot Tubs, Locus Coeruleus Modulation and Hel-
4. Zhang Y, Talalay P, Cho CG, Posner GH (1992) A major inducer of anticarcinogenic minth Therapy. American College of Neuropsychopharmacology 52ndAnnual Meeting
protective enzymes from broccoli: Isolation and elucidation of structure. Proc Natl (American College of Neuropsychopharmacology) Dec. 12, 2013, Hollywood, FL.
Acad Sci USA 89(6):2399–2403. 25. Wiggins JL, et al. (2011) Using a self-organizing map algorithm to detect age-related
5. Talalay P (2000) Chemoprotection against cancer by induction of phase 2 enzymes. changes in functional connectivity during rest in autism spectrum disorders. Brain Res
Biofactors 12(1-4):5–11. 1380:187–197.
6. Juge N, Mithen RF, Traka M (2007) Molecular basis for chemoprevention by 26. Gan N, et al. (2010) Sulforaphane activates heat shock response and enhances pro-
teasome activity through up-regulation of Hsp27. J Biol Chem 285(46):35528–35536.
sulforaphane: A comprehensive review. Cell Mol Life Sci 64(9):1105–1127.
27. Marshburn EC, Aman MG (1992) Factor validity and norms for the aberrant behavior
7. Baird L, Dinkova-Kostova AT (2011) The cytoprotective role of the Keap1-Nrf2 path-
checklist in a community sample of children with mental retardation. J Autism Dev
way. Arch Toxicol 85(4):241–272.
Disord 22(3):357–373.
8. Egner PA, et al. (2011) Bioavailability of Sulforaphane from two broccoli sprout
28. Constantino JN, et al. (2003) Validation of a brief quantitative measure of autistic
beverages: Results of a short-term, cross-over clinical trial in Qidong, China. Cancer
traits: Comparison of the social responsiveness scale with the autism diagnostic in-
Prev Res (Phila) 4(3):384–395.
terview-revised. J Autism Dev Disord 33(4):427–433.
9. Dinkova-Kostova AT (2012) The role of sulfhydryl reactivity of small molecules for the
29. Choque Olsson N, Bölte S (2014) Brief report: “Quick and (not so) dirty” assessment
activation of the KEAP1/NRF2 pathway and the heat shock response. Scientifica
of change in autism: Cross-cultural reliability of the Developmental Disabilities CGAS
(Cairo) 2012:606104.
and the OSU autism CGI. J Autism Dev Disord 44(7):1773–1778.

MEDICAL SCIENCES
10. Kensler TW, et al. (2013) Keap1-nrf2 signaling: a target for cancer prevention by
30. The OSU Research Unit on Pediatric Psychopharmacology (2005) OSU Autism Rating
sulforaphane. Top Curr Chem 329:163–177.
Scale (Ohio State Univ, Columbus, OH).
11. James SJ, et al. (2004) Metabolic biomarkers of increased oxidative stress and im-
31. Deykin EY, MacMahon B (1979) The incidence of seizures among children with autistic
paired methylation capacity in children with autism. Am J Clin Nutr 80(6):1611–1617.
symptoms. Am J Psychiatry 136(10):1310–1312.
12. James SJ, et al. (2009) Cellular and mitochondrial glutathione redox imbalance in
32. Danielsson S, Gillberg IC, Billstedt E, Gillberg C, Olsson I (2005) Epilepsy in young
lymphoblastoid cells derived from children with autism. FASEB J 23(8):2374–2383. adults with autism: A prospective population-based follow-up study of 120 in-
13. Giulivi C, et al. (2010) Mitochondrial dysfunction in autism. JAMA 304(21):2389–2396. dividuals diagnosed in childhood. Epilepsia 46(6):918–923.
14. Napoli E, Wong S, Hertz-Picciotto I, Giulivi C (2014) Deficits in bioenergetics and im- 33. Miles JH (2010) Workshop Report: Fever and Autism. Available at http://sfari.org/news-
paired immune response in granulocytes from children with autism. Pediatrics 133: and-opinion/workshop-reports/2010/workshop-report-fever-and-autism. Accessed Au-
e1405–e1410. gust 20, 2014.
15. Frackowiak J, Mazur-Kolecka B, Schanen NC, Brown WT, Wegiel J (2013) The link 34. Hochberg Z, et al. (2011) Child health, developmental plasticity, and epigenetic
between intraneuronal N-truncated amyloid-β peptide and oxidatively modified lip- programming. Endocr Rev 32(2):159–224.
ids in idiopathic autism and dup(15q11.2-q13)/autism. Acta Neuropathol Commun 35. Rangasamy S, D’Mello SR, Narayanan V (2013) Epigenetics, autism spectrum, and
1(1):61. neurodevelopmental disorders. Neurotherapeutics 10(4):742–756.
16. Vargas DL, Nascimbene C, Krishnan C, Zimmerman AW, Pardo CA (2005) Neuroglial 36. Egner PA, et al. (2014) Rapid and sustainable detoxication of airborne pollutants by
activation and neuroinflammation in the brain of patients with autism. Ann Neurol broccoli sprout beverage: Results of a randomized clinical trial in china. Cancer Prev
57(1):67–81. Res (Phila) 7(8):813–823.
17. Frye RE, Rossignol DA (2014) Treatments for biomedical abnormalities associated 37. Lyall K, Schmidt RJ, Hertz-Picciotto I (2014) Maternal lifestyle and environmental risk
with autism spectrum disorder. Frontiers Pediatrics 2(66):1–8. factors for autism spectrum disorders. Int J Epidemiol 43(2):443–464.
18. Brose RD, et al. (2012) Activation of the stress proteome as a mechanism for small 38. Constantino JN, et al. (2009) Developmental course of autistic social impairment in
molecule therapeutics. Hum Mol Genet 21(19):4237–4252. males. Dev Psychopathol 21(1):127–138.
19. Fahey JW, Talalay P, Kensler TW (2012) Notes from the field: “Green” chemo- 39. Liang KY, Zeger S (2000) Longitudinal data analysis of continuous and discrete re-
prevention as frugal medicine. Cancer Prev Res (Phila) 5(2):179–188. sponses for pre–post designs. Sankhya : The Indian Journal of Statistics 62(1):134–148.

Singh et al. PNAS | October 28, 2014 | vol. 111 | no. 43 | 15555
Supporting Information
Singh et al. 10.1073/pnas.1416940111
SI Materials and Methods time point for each participant were analyzed. The four partic-
Preparation of Sulforaphane-Rich Broccoli Sprout Extracts. Sulforaphane- ipants who discontinued early were not included in the main
rich broccoli sprout extract (SF-BSE) was prepared by the Cullman analyses.
Chemoprotection Center at The Johns Hopkins University essen- An alternative, intention-to-treat (ITT) analysis included all
tially as described in Egner et al. (1). In brief, specially selected randomized participants. Each outcome was modeled in a mixed-
broccoli seeds were surface-disinfected and grown (sprouted) for effects general linear model with fixed effects for visit and the
3 d in a commercial sprouting facility under controlled light and interaction of postrandomization visit and treatment group and
moisture conditions. A boiling water extract was prepared, filtered, random participant-specific intercepts and slopes with un-
cooled, and treated with the enzyme myrosinase (from daikon structured covariance. The absence of a main effect for treatment
sprouts) to convert precursor glucosinolates to isothiocyanates, and (i.e., a “shared baseline”) properly reflects the true state of the
then lyophilized at a food processing facility (Oregon Freeze Dry, population sampled before randomization and has the advantage
Albany, OR). The lyophilized powder (216 μmol SF/g powder) was of adjusting for any chance differences at baseline in a manner
encapsulated into #1 gelcaps by ALFA Specialty Pharmacy (Co- similar to ANCOVA. The model was used to estimate the dif-
lumbia, MD); each capsule contained 50 μmol SF (232 mg of SF- ference between treatment and control in mean change from
BSE); placebo capsules were filled with microcrystalline cellulose. baseline at each time point. Given its assumptions, the mixed
The powders (bulk and capsules) were maintained at approximately model yields estimates that are unbiased as long as missing test
−20 °C and repeatedly checked for microbial contaminants and SF scores are predictable from observed scores. In this model, the
titer before conveyance to the study site pharmacy (Massachusetts baseline scores (the mean of scores at the screening and ran-
General Hospital) to be dispensed to patients. domization visits) of the four participants lost to follow-up were
included in the analysis, with all participants analyzed according
Safety and Tolerance. Adverse event monitoring and documenta- to their assigned treatment group without regard to whether they
tion by severity, duration, and relatedness were performed by continued treatment. The test statistic used was the difference
a physician at each follow-up visit. Study drug pause and stop rules between the two treatment groups in the average change in SRS
were formulated as follows: In case of any significant adverse from baseline to 18 wk. We used a two-tailed test at α = 0.05. To
events or if laboratory values were above the study eligibility rules quantify the change in efficacy after 4 wk without treatment we
[i.e., alanine transaminase or aspartate transaminase > 1.5× upper estimated the difference in the treatment-placebo comparison at
limit of normal, serum creatinine > 1.2 mg/dL or thyroid stimu- 22 vs. 18 wk, by a simple linear contrast of the estimated model
lating hormone (TSH) outside normal limits], study medication parameters.
was to be stopped for 2 wk and subjects reevaluated. If laboratory By mixed model analysis of the ITT sample, the sulforaphane
studies returned to normal and no more adverse events were group had significantly greater improvement in their overall SRS
noted, the medication was to be restarted. Otherwise, the study and ABC scores compared with the placebo group. After 18 wk
medication was to be stopped indefinitely. Study drug safety and of treatment, the sulforaphane group had a reduction in SRS total
adherence to the protocol were monitored at quarterly meetings score of 15.3 units from baseline compared with a decrease of
of a Data Safety Monitoring Board constituted by three members 3.2 units in the placebo group (difference = 12.1 units, P = 0.01).
at the Lurie Center for Autism. After subjects stopped the medication, the SRS scores tended to
revert to the mean (although incompletely), with the average
Quality of Data Certification. An on-site Food and Drug Admin- decrease of SRS total score from baseline to 22 wk being 6 units
istration Good Clinical Practice Raw Data Audit was performed in the sulforaphane group and 2.9 units in the placebo group
on May 19–21, 2014. The inspection was performed by Philippe (difference = 3.1 units, P = 0.70).
Ourisson (Manager, Quality Assurance and Scientific Support), The effect of sulforaphane on ABC total scores was compa-
and confirmed by Paul Swidersky (President), Quality Associates, rable, with a decrease of 17.6 units among participants ran-
Fulton, MD. domized to active treatment compared with a decrease of 0.08
The folder of each of the 44 subjects was reviewed. Every in- unit in the placebo group (difference = 17.5 units, P = 0.0001). As
formed consent form was examined. All inclusion/exclusion criteria with SRS scores, ABC total scores also tended to revert to
were verified. Most baseline physicals and all subject evaluations baseline after stopping treatment, such that the average decrease
through week 22 were examined, then transcribed to a spreadsheet. of total ABC score from baseline to 22 wk was 8.8 units in the
No evidence was obtained that there were significant errors in sulforaphane group, compared with 0.08 unit in the placebo
collecting the data and transferring the results to the spreadsheets. group (difference = 8.9 units, P = 0.28).
Guided by the power analysis and sample size calculation
Statistical Analysis of Outcome Measures and Intention-to-Treat discussed in the main text (indicating power to detect a 15-point
Analysis. Our primary hypotheses concerned differences between change in total SRS score), a positive response to treatment was
the sulforaphane and placebo treatment groups in the average defined post hoc as a 30% decrease from baseline on SRS and
change in Abberrant Behavior Checklist (ABC) and Social Re- ABC scores. After conservatively considering the four dropouts
sponsiveness Scale (SRS) scores from baseline to 18 wk, and their (one on placebo and three on sulforaphane) as not meeting the
reversion to baseline at 22 wk. Clinical Global Impression Im- 30% reduction threshold, ABC scores at 18 wk were available for
provement Scale (CGI-I) scores were examined as a secondary 39 participants (11 placebo and 28 sulforaphane), and SRS scores
outcome at the same time points. at 18 wk were available for 41 participants (12 placebo and 29
Of the 44 subjects originally enrolled and randomized to sul- sulforaphane). Additionally, three participants on sulforaphane
foraphane treatment (n = 29) or placebo (n = 15), 4 subjects inexplicably had >30% reduction in their ABC scores from
discontinued participation in the study before the first return baseline, whereas they were not improved either on CGI-I
visit, 4 wk after treatment initiation. In the main text, change (OACIS-I) or poststudy parent interviews. These three partic-
scores calculated as the change from baseline to each follow-up ipants were conservatively considered as not meeting the 30%

Singh et al. www.pnas.org/cgi/content/short/1416940111 1 of 7


reduction threshold. With that adjustment, 31% (9 of 29) par- were “much improved” or “very much improved” on the aberrant
ticipants on sulforaphane had a positive response on SRS com- or abnormal behavior subdomain of the CGI-I compared with
pared with 0% (0 of 15) participants on placebo (Fisher’s exact 6.7% (1 of 15) of placebo participants (P = 0.007). Thirty-eight
test, P = 0.018). Similarly, 44.8% (13 of 29) of participants taking percent (11 of 29) participants on sulforaphane were “much im-
sulforaphane had a positive response on ABC compared with proved” or “very much improved” on the verbal communication
13% (2 of 15) patients on placebo (P = 0.048). subdomain of the CGI-I compared with 6.7% (1 of 15) of placebo
Also consistent with the “per-protocol” findings reported in
participants (P = 0.035).
the main text, an ITT analysis suggested that more participants
In conclusion, the ITT data analysis is presented as the most
in the sulforaphane compared with the placebo group experi-
enced a positive response on several subdomains of the CGI-I conservative approach to evaluation of the data, and results in
scale. After 18 wk of treatment, 41% (12 of 29) participants similar findings that confirm the per-protocol analysis. Moreover,
taking sulforaphane were “much improved” or “very much im- whether those participants who dropped out of the study are
proved” on the social interaction subdomain of the CGI-I com- included or excluded does not detract from the statistical validity
pared with 0% (0 of 15) of participants on placebo (P = 0.003). of sulforaphane’s effects in this clinical trial of young men with
Forty-eight percent (14 of 29) of participants on sulforaphane autism spectrum disorder.

1. Egner PA, et al. (2014) Rapid and sustainable detoxication of airborne pollutants by
broccoli sprout beverage: Results of a randomized clinical trial in China. Cancer Prev
Res (Phila) 7(8):813–823.

Singh et al. www.pnas.org/cgi/content/short/1416940111 2 of 7


Fig. S1. Flow diagram of progress through phases of the study.

Singh et al. www.pnas.org/cgi/content/short/1416940111 3 of 7


Table S1. Baseline characteristics of patients (n = 44) who volunteered for the study
Characteristics Placebo group (n = 15) Sulforaphane group (n = 29) P value*

Age (Mean ± SD) 16.6 ± 3.5 17.9 ± 3.9 0.27


Weight (lbs.) 154.7 ± 39.7 170.9 ± 50.4 0.28
Body mass index 23.7 ± 5.1 25.9 ± 6.5 0.26
Head circumference (cm) 56.3 ± 2.8 58.1 ± 2.4 0.06
Pulse rate per minute 88 ± 15 80 ± 14 0.07
Temperature (° F) 98.6 ± 0.6 98.4 ± 0.9 0.27
Systolic blood pressure (mm Hg) 117 ± 13 119 ± 14 0.55
Diastolic blood pressure (mm Hg) 73 ± 6 76 ± 8 0.31
Abnormal physical examination findings
Skin 1/15 4/29 0.65
Head, eye, ear, nose, throat 1/15 1/29 1.0
Neck/thyroid 0/15 0/29 —
Chest/lungs 1/15 0/29 1
Cardiovascular 1/15 0/29 0.33
Abdominal 0/15 0/29 —
Neurological 1/15 2/29 1.0
Dysmorphic features 1/15 3/29 1.0
Race/ethnicity
White 13/15 26/29 0.5
Black 0/15 2/29
Hispanic 1/15 0/29
Asian 1/15 1/29
Reported history of fever effects 11/15 23/29
Autism Diagnostic Observation Schedule score
Communication 6.0 ± 1.9 6.6 ± 2.0 0.36
Social interaction 11.6 ± 2.0 11.2 ± 1.7 0.50
Communication + social interaction 17.6 ± 3.1 17.8 ± 3.1 0.86
Stereotyped behavior and restricted interests 3.6 ± 1.7 2.9 ± 1.5 0.16
ABC score (screening and baseline averaged)
Total raw score 60.0 ± 23.2 63.6 ± 25.3 0.65
Irritability subscale 14.2 ± 8.9 13.7 ± 9.3 0.89
Lethargy subscale 13.9 ± 6.3 15.1 ± 7.6 0.62
Stereotypy subscale 10.3 ± 5.2 10.1 ± 4.5 0.93
Hyperactivity subscale 18.2 ± 8.5 19.7 ± 9.3 0.59
Inappropriate speech subscale
SRS score (screening and baseline averaged)
Total raw score 120.1 ± 16.6 122.2 ± 24.1 0.77
Awareness subscale 14.6 ± 2.8 15.8 ± 3.8 0.30
Cognition subscale 21.0 ± 3.4 23.2 ± 5.5 0.16
Communication subscale 41.5 ± 7.1 40.9 ± 8.6 0.84
Motivation subscale 18.4 ± 4.0 19.2 ± 4.9 0.60
Mannerisms subscale 25.2 ± 4.8 23.3 ± 6.3 0.31
Ohio Autism Clinical Global Impression Severity
Scale-severity score
General level of autism 4.53 ± 0.74 4.38 ± 0.56 0.45
Social interaction 4.80 ± 1.01 4.51 ± 0.69 0.28
Aberrant/abnormal behavior 4.20 ± 1.37 4.21 ± 0.86 0.99
Repetitive/ritualistic behavior 4.13 ± 0.83 4.14 ± 0.74 0.99
Verbal communication 4.53 ± 1.36 4.45 ± 0.95 0.81
Nonverbal communication 4.27 ± 0.96 4.10 ± 0.72 0.53
Hyperactivity and inattention 4.40 ± 0.91 4.10 ± 0.90 0.31
Anxiety 4.33 ± 1.23 4.17 ± 0.71 0.65
Sensory sensitivities 4.40 ± 0.74 4.07 ± 0.65 0.13
Restricted and narrow interests 4.33 ± 0.72 4.41 ± 0.63 0.70
Hematology laboratories
Hematocrit (ref: 37.5–51.0%) 43.7 ± 2.3 44.2 ± 2.3 0.56
Hemoglobin (ref: 12.6–17.7 g/dL) 14.9 ± 0.8 15.2 ± 0.9 0.46
Mean corpuscular hemoglobin (ref: 26.6–33.0 pg) 29.7 ± 1.6 29.67 ± 1.5 0.89
Mean corpuscular hemoglobin concentration 34.1 ± 0.7 34.6 ± 0.7 0.06
(ref: 31.5–35.7 g/dL)
Mean corpuscular volume (ref: 79–97 fL) 86.4 ± 3.2 85.6 ± 4.2 0.54
Platelet count (ref: 150–349 × 103/μL) 245.5 ± 47.8 251.5 ± 55.5 0.74
RBC count (ref: 4.14–5.80 × 106/μL) 5.1 ± 0.3 5.2 ± 0.5 0.27

Singh et al. www.pnas.org/cgi/content/short/1416940111 4 of 7


Table S1. Cont.
Characteristics Placebo group (n = 15) Sulforaphane group (n = 29) P value*

WBC count (ref: 4.0–9.1 × 10 /μL)3


6.9 ± 2.3 6.8 ± 1.7 0.85
Blood chemistry laboratories
Sodium (mEq/L) (ref: 135–145 mmol/L) 139.4 ± 3.4 139.8 ± 2.2 0.68
Potassium (ref: 3.4–4.8 mmol/L) 3.7 ± 0.5 3.8 ± 0.3 0.72
Chloride (ref: 100–108 mmol/L) 105 ± 3.1 105 ± 2.4 0.97
CO2 (ref: 23.0–31.9 mmol/L) 23.6 ± 2.4 22.3 ± 3.2 0.17
Anion gap 10.9 ± 3.5 12.5 ± 3.0 0.13
Blood urea nitrogen (BUN, ref: 8–25 mg/dL) 14 ± 5.1 12.7 ± 3.1 0.41
Serum creatinine (ref: 0.60–1.50 mg/dL) 0.8 ± 0.1 0.8 ± 0.1 0.85
Serum glutamic oxaloacetic transaminase 22.8 ± 6.4 22.6 ± 8.6 0.92
(SGOT, ref: 10–40 U/L)
Serum glutamic pyruvic transaminase 18.3 ± 6.1 30.0 ± 20.6 0.009
(SGPT, ref: 10–55 U/L)
Alkaline phosphatase (ref: 15–350 U/L) 180.9 ± 122.1 116.4 ± 50.8 0.08
Total bilirubin (ref: 0.0–1.0 mg/dL) 0.6 ± 0.2 0.4 ± 0.3 0.12
TSH (ref: 0.5–4.0 μIU/mL) 1.6 ± 0.9 2.0 ± 2.1 0.35
Abnormal urinalysis results
Urine bilirubin 0/12 0/22 —
Urine glucose 0/12 0/22 —
Urine ketones 0/12 1/22 1.0
Urine occult blood 0/12 0/22 —
Urine protein 1/12 2/22 1.0
Urine pH 6.7 ± 0.8 7.0 ± 0.8 0.28

*P values are based on two-tailed t tests, Fisher’s exact, or χ2 tests.

Singh et al. www.pnas.org/cgi/content/short/1416940111 5 of 7


Table S2. Summary of adverse events reported and safety laboratories at 18 wk
Clinical indicators Placebo group (n = 14) Sulforaphane group (n = 26) P value*

Weight gain (lbs.) 0.31 ± 6.16 4.31 ± 5.87 0.05


Heart rate (beats per minute) 88 ± 15 79 ± 13 0.06
Temperature (° F) 98.6 ± 0.87 98.3 ± 0.81 0.28
Vomiting 1 (7.1%) 5 (19.2%) 0.40
Aggressions 2 (14.3%) 4 (15.4%) 1.00
Abdominal pain 2 (14.3%) 4 (15.4%) 1.00
Flatulence 2 (14.3%) 4 (15.4%) 1.00
Irritability 0 (0%) 3 (11.5%) 0.54
Constipation 2 (14.3%) 3 (11.5%) 1.00
Diarrhea 1 (7.1%) 3 (11.5%) 1.00
Fever 1 (7.1%) 3 (11.5%) 1.00
Headache 0 (0%) 3 (11.5%) 0.53
Allergy exacerbation 0 (0%) 3 (11.5%) 0.54
Unprovoked seizures 0 (0%) 2 (7.7%) 0.53
Stubbornness 0 (0%) 2 (7.7%) 0.53
Insomnia 4 (28.6%) 2 (7.7%) 0.16
Cough 0 (0%) 2 (7.7%) 0.53
Agitation 1 (7.1%) 1 (3.8%) 1.00
Crying spells 1 (7.1%) 1 (3.8%) 1.00
Hyperactivity 2 (14.3%) 1 (3.8%) 0.28
Obsessive compulsive disorder 0 (0%) 1 (3.8%) 1.00
Impatience 0 (0%) 1 (3.8%) 1.00
Fidgety 0 (0%) 1 (3.8%) 1.00
Disinhibition 0 (0%) 1 (3.8%) 1.00
Self-injurious behavior 0 (0%) 1 (3.8%) 1.00
Pica 0 (0%) 1 (3.8%) 1.00
Increased appetite 1 (7.1%) 1 (3.8%) 1.00
Mouth sores 0 (0%) 1 (3.8%) 1.00
Daytime sleepiness 0 (0%) 1 (3.8%) 1.00
Sore throat 0 (0%) 1 (3.8%) 1.00
Asthma exacerbation 0 (0%) 1 (3.8%) 1.00
Anxiety 2 (14.3%) 0 (0%) 0.12
Lethargy 2 (14.3%) 0 (0%) 0.12
Burping 3 (21.4%) 0 (0%) 0.03
Decreased appetite 1 (7.1%) 0 (0%) 0.35
Increased urination 1 (7.1%) 0 (0%) 0.35
Hematology labs at 18 wk
Hematocrit (ref: 37.5–51.0%) 43.4 ± 3.5 43.4 ± 2.5 0.99
Hemoglobin (ref: 12.6–17.7 g/dL) 14.7 ± 1.3 14.9 ± 1.0 0.51
Mean corpuscular hemoglobin (ref: 26.6–33.0 pg) 29.4 ± 1.2 29.7 ± 1.6 0.59
Mean corpuscular hemogobin concentration 33.9 ± 0.6 34.4 ± 0.8 0.09
(ref: 31.5–35.7 g/dL)
Mean corpuscular volume (ref: 79–97 fL) 86.6 ± 3.4 86.0 ± 3.8 0.60
Platelet count (ref: 150–349 × 103/μL) 236.8 ± 44.8 237.1 ± 45.2 0.98
RBC count (ref: 4.14–5.80 × 106/μL) 5.0 ± 0.4 5.1 ± 0.4 0.53
WBC count (ref: 4.0–9.1 × 103/μL) 6.3 ± 1.7 5.9 ± 1.6 0.50
Abnormal Hematology laboratories
(outside of reference range)
Hematocrit 0/13 0/25
Hemoglobin 0/13 0/25
Mean corpuscular hemoglobin 0/13 1/25 1.00
Mean corpuscular hemoglobin concentration 0/13 1/25 1.00
Mean corpuscular volume 0/13 1/25 1.00
Platelet count 1/13 1/25 0.57
RBC count 0/13 1/25 1.00
WBC count 1/13 2/25 1.00
Blood chemistry labs at 18 wk
Sodium (ref: 135–145 mmol/L) 139.8 ± 1.7 139.7 ± 1.9 0.83
Potassium (ref: 3.4–4.8 mmol/L) 3.6 ± 0.4 3.8 ± 0.3 0.30
Chloride (ref: 100–108 mmol/L) 103.9 ± 2.5 103.5 ± 2.3 0.64
CO2 (ref: 23.0–31.9 mmol/L) 23.6 ± 2.0 24.2 ± 2.6 0.51
Anion gap 12.3 ± 2.9 12.0 ± 2.2 0.48
BUN (ref: 8–25 mg/dL) 12.3 ± 2.8 13.0 ± 3.4 0.57

Singh et al. www.pnas.org/cgi/content/short/1416940111 6 of 7


Table S2. Cont.
Clinical indicators Placebo group (n = 14) Sulforaphane group (n = 26) P value*

Serum creatinine (ref: 0.60–1.50 mg/dL) 0.8 ± 0.1 0.8 ± 0.1 0.34
SGOT (ref: 10–40 U/L) 22.5 ± 9.2 22.5 ± 5.4 0.98
SGPT (ref: 10–55 U/L) 22.2 ± 20.1 31.1 ± 20.7 0.22
Alkaline phosphatase (ref: 15–350 U/L) 136.9 ± 52.5 112.5 ± 53.2 0.19
Total bilirubin (ref: 0.0–1.0 mg/dL) 0.6 ± 0.3 0.5 ± 0.3 0.54
TSH (ref: 0.5–4.0 μIU/mL) 1.5 ± 0.6 1.9 ± 1.1 0.19
Abnormal blood chemistry laboratories (outside
of reference range)
SGPT 1/13 4/24 0.64
CO2 5/13 6/24 0.46
Sodium 0/13 1/24 1.00
Potassium 2/13 1/24 0.28
Chloride 1/13 1/24 1.00
BUN 1/13 1/24 1.00
Serum creatinine 0/13 0/24 —
SGOT 1/13 0/24 0.35
Alkaline phosphatase 0/13 0/24 —
Total bilirubin 1/13 2/24 1.00
TSH 1/13 1/24 1.00
Urinalysis at 18 wk —
Urine bilirubin 0/13 0/24 —
Urine glucose 0/13 1/24 1.00
Urine ketones 0/13 1/24 1.00
Urine occult blood 0/13 0/24 —
Urine protein 1/13 0/24 0.35
Urine pH (ref: 5.0–7.5) 6.8 ± 0.5 7.2 ± 0.8 0.03
Urine pH outside of reference range 1/13 7/24 0.22

*P values are based on two-tailed t tests, Fisher’s exact, or χ2 tests.

Dataset S1. Complete safety laboratory studies (hematology, chemistry, urinalysis)

Dataset S1

Singh et al. www.pnas.org/cgi/content/short/1416940111 7 of 7