Am. J. Trop. Med. Hyg., 91(4), 2014, pp. 777–785
doi:10.4269/ajtmh.14-0093
Copyright © 2014 by The American Society of Tropical Medicine and Hygiene
Short-Term Safety and Efficacy of Calcium Montmorillonite Clay (UPSN) in Children
Nicole J. Mitchell, Justice Kumi, Mildred Aleser, Sarah E. Elmore, Kristal A. Rychlik, Katherine E. Zychowski,
Amelia A. Romoser, Timothy D. Phillips, and Nii-Ayi Ankrah*
College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas; Noguchi Memorial Institute
for Medical Research, University of Ghana, Legon, Accra, Ghana; Ejura-Sekyedumase District Hospital, Ejura, Ghana
Abstract. Recently, an association between childhood growth stunting and aflatoxin (AF) exposure has been identi-
fied. In Ghana, homemade nutritional supplements often consist of AF-prone commodities. In this study, children were
enrolled in a clinical intervention trial to determine the safety and efficacy of Uniform Particle Size NovaSil (UPSN),
a refined calcium montmorillonite known to be safe in adults. Participants ingested 0.75 or 1.5 g UPSN or 1.5 g calcium
carbonate placebo per day for 14 days. Hematological and serum biochemistry parameters in the UPSN groups were
not significantly different from the placebo-controlled group. Importantly, there were no adverse events attributable to
UPSN treatment. A significant reduction in urinary metabolite (AFM1) was observed in the high-dose group compared
with placebo. Results indicate that UPSN is safe for children at doses up to 1.5 g/day for a period of 2 weeks and can
reduce exposure to AFs, resulting in increased quality and efficacy of contaminated foods.
INTRODUCTION with lower weight-for-age and height-for-age z scores. Fur-
thermore, children ages 16–37 months from Benin exhibited
Stunting, wasting, and fetal growth retardation result in a significant negative correlation between AF exposure and
more than 2 million deaths in children under the age of height increase over 8 months.3 The growth stunting observed
5 years and account for 21% of disability-adjusted life years in children from Benin and Togo had a distinct dose-response
worldwide.1 In 2010, it was estimated that 171 million pre- relationship with AF biomarker levels in the serum (AF-alb).
school children worldwide were stunted.2 Although the Children with a height-for-age z score of £ −2 had 30–40%
overall prevalence of stunting in developing countries has higher AF-alb compared with children not classified as
improved over the past two decades (from 44.4% to 29.2%), stunted.4,5 Aside from its effects on growth, AF is also a
the majority of this improvement has been occurring in Asia potent carcinogen and can have deleterious effects on immune
and Latin America. The rate in Africa (40%), however, has status and liver health.7,20–24 Although the exact mechanisms
remained stagnant and is not expected to improve drastically of AF-induced growth stunting are not well-defined at this
over the next 10 years.2 Although stunting is primarily attrib- time, animal studies have indicated that AF exposure results
uted to nutritional and protein deficiencies, aflatoxin (AF), a in decreased weight gain, lowered feed conversion efficiency,
common maize and peanut contaminant in Africa and Asia, vitamin depletion, and inhibited protein synthesis.25–35 As a
has also been associated with growth faltering in sub-Saharan result of these epidemiological studies, it is clear that inter-
Africa.3–10 Current strategies implemented to alleviate growth vention strategies directed at the mitigation of child exposures
faltering and subsequent physical and mental deficits include are needed in areas where risk of AF consumption is high
food and micronutrient supplementation. These complemen- and malnutrition is common.
tary foods are designed to supplement the typical diet and Inclusion of a calcium montmorillonite clay, NovaSil (NS),
often consist primarily of maize and groundnuts (peanuts), in animal feed has shown efficacy in reducing bioavailability
putting children at risk for AF exposure.11–13 Recently, the of AFB after ingestion 36–381 . For example, young broiler chicks
population in which this trial was carried out was determined administered 5 ppm AFB1 and 0.5% (wt/wt) NS clay were
to be consuming homemade Weanimix with average contami- protected from the growth inhibitory effects of AF observed
nation levels exceeding 200 ppb Aflatoxin B1 (AFB1) and some in controls.34 In growing barrows, NS prevented toxicity of
containing up to 500 ppb.14 Because of dietary exposures in AF over a 4-week period, which was determined by recovery
African countries, such as Guinea, Kenya, Benin, Togo, Sene- of weight gain and serum alkaline phosphatase (ALP) and
gal, and The Gambia, approximately 85–100% of children have g-glutamyl transferase (GGT) values compared with control
detectable levels of serum or urinary AF biomarkers.3,5,7,15–19 group levels.39,40 In addition, NS has also been shown to
Increasing evidence that children in sub-Saharan Africa significantly reduce AF biomarkers of exposure, including
are chronically exposed to AF has resulted in recent efforts urinary metabolite (AFM1) in urine and milk and AF-alb
to understand the role that AF plays in growth stunting and in serum of various animal models.38,41–43 Human interven-
its influence on morbidity and mortality rates. Turner and tion trials with NS products in Texas and Ghana have also
others6 followed the growth of 138 Gambian infants from reported a similar reduction of AF bioavailability, resulting
birth to 12 months and compared growth status with mater- in as much as a 58% decrease in biomarker levels.44,45
nal AF exposure during pregnancy. In this study, a higher Recently, the NS parent compound was refined, produc-
mean maternal exposure level was significantly correlated ing a product that is more uniform in particle size, with the
majority of the particles residing in the 45- to 100-mm range.
The new product, Uniform Particle Size NovaSil (UPSN),
displayed a similar AFB1 sorption capacity to that of NS
* during isothermal analysis and was deemed to be more palat-Address correspondence to Nii-Ayi Ankrah, Noguchi Memorial
able for human consumption.46Institute for Medical Research, University of Ghana, PO Box LG In vivo efficacy of UPSN was
581, Legon, Accra, Ghana. E-mail: NAnkrah@noguchi.ug.edu.gh verified in Ghanaian adults during a crossover-designed
777
778 MITCHELL AND OTHERS
trial, in which UPSN was added to participants’ food before the placebo group was used as a reference when determin-
ingestion.45 Results from mineralogical analyses of UPSN ing safety of UPSN. Doses were weighed into identical
and NS indicated similar structural, morphological, and packages at the Noguchi Memorial Institute for Medical
chemical characteristics; therefore, the two materials are Research (NMIMR) before the study to ensure that moni-
thought to possess similar safety profiles.46 tors and participants would be blinded to their treatment.
Safety assessment of both NS and UPSN has been conducted Trained study monitors mixed each participant’s treatment
in animal and adult human trials.36,46–48 Results of NS clay into their normal breakfast and dinner meals provided by
phase I and II clinical trials suggest that ingestion of up to 3 g/ the participant’s parents. No additional foods were provided
day in adults is safe for a 3-month period.49–51 Based on by the clinical team. Breakfast meals typically consisted of a
detailed studies conducted in animals and humans, it was deter- corn-based porridge called koko or soup, and the dinner
mined that ingestion of UPSN at levels efficacious for reducing meals typically consisted of a common soup (i.e., peanut
AFB1 biomarkers would be reasonably safe in children. soup or lamb lite soup) and corn or cassava dough called
In this clinical trial, safety and efficacy of UPSN were banku and fufu, respectively. These meals were supplied by
assessed for children at risk for AF exposure from the Ejura- the individual households. Participants provided blood sam-
Sykedumase district of Ghana. The study followed a double- ples (3 mL) on the morning before initiation of treatment
blind, placebo-controlled trial design over a 2-week time (day 0) and day 15 (the morning after their last treatment
period. The results from this research will be used to design dose). Blood samples were collected by trained phleboto-
future studies investigating long-term protection of children mists at the Ejura District Hospital. Aliquots of the blood
at high risk for AF exposure and the potential of this material samples were used for hematological analysis, and the
for short-term therapy during outbreaks of acute aflatoxicosis. remaining amount was centrifuged. The resulting serum was
collected and kept at −20°C. Urine samples were collected by
MATERIALS AND METHODS parents on the morning of day 0 (baseline), halfway through
the study (day 7), and the morning after the final dose (day
Materials. UPSN was obtained from BASF (Jackson, MS). 15). After collection, urine samples were stored at −20°C and
UPSN was examined for various environmental contami- together with the serum samples, transported to NMIMR for
nants, including dioxins and heavy metals, to ensure compli- analysis. The study design followed the guidelines for a double-
ance with federal and international standards. Metal and blind randomized clinical trial. Ethical clearance and Institu-
dioxin analyses of both NS (BASF) and UPSN were reported tional Review Board approval for this study were obtained
to be similar and well under the tolerable daily intake (TDI) from both Texas A&M University and NMIMR (2011-0684
or provisional tolerable daily intake (PTDI) set forth by the and 043/11-12, respectively).
World Health Organization (WHO) and the Joint Food and Adverse events monitoring. Based on the existing scientific
Agriculture Organization/WHO Expert Committee on Food literature describing consumption of dioctahedral smectite
Additives (JECFA).46 UPSN was sterilized by electron beam clays in adults and children, no severe toxicity was expected
irradiation to prevent any possible bacterial or viral contamina- as a result of UPSN treatment. However, research staff and
tion before trial initiation. medical personnel were on site throughout the study period
High-performance liquid chromatography-grade methanol, to monitor for potential adverse effects and remove partici-
phosphate-buffered saline, and AFM1 standard were pur- pants from the study in the event of such an effect. Daily
chased from Sigma Aldrich (Saint Louis, MO). Ultrapure diary worksheets and symptom checklists were provided to
deionized water (18.2 MW) was generated within the labora- study monitors as assessment tools for adverse events moni-
tory using an Elga Automated Filtration System (Woodridge, toring and completed two times daily after ingestion of each
IL). Immunoaffinity columns were purchased from VICAM treatment dose. Adverse events are described as percentages
(Milford, MA). of the total numbers of adverse event reports out of the total
Study site and participants. Study participants were recruited numbers of completed daily diary worksheets per treatment
from six communities in the Ejura-Sekyedumase district of group. In the event of an adverse treatment effect or unrelated
the Ashanti Region of Ghana. The six communities included condition at any time during the study, medical treatment was
Dromonkuma, Hiawoanwu, Kotokoliline, Nkwanta, Ejurafie, available to participants from the district hospital at no cost
and Kasei. These communities are in rural areas, where to the participant. Any symptoms were assessed according to
inhabitants are primarily subsistence farmers. All recruited the following criteria: mild (grade 1), slightly bothersome and
participants were between 3 and 9 years of age. Consent relieved with symptomatic treatment; moderate (grade 2),
was sought from the parents or legal guardians after a com- bothersome and interfered with activities and only partially
munity meeting with study personnel. Consent documents relieved with symptomatic treatment; severe (grade 3), pre-
were translated and explained to each participant, and then vented regular activities and not relieved with symptomatic
signed by each participant’s guardian before initiation of the treatment. Any participant experiencing a severe symptom
study. Participants were randomly assigned to one of three was advised to seek immediate medical attention. Physical
treatment groups. The three treatment arms consisted of a examination and laboratory analysis were performed for
placebo group, which received 0.75 g calcium carbonate two persistent symptoms. Any symptoms that were linked to the
times daily, a low-dose group, which received 0.375 g UPSN UPSN treatment by the study physician would result in imme-
two times daily, and a high-dose group, which received diate discontinuation of the treatment; however, this did not
0.75 g UPSN two times daily. A placebo-controlled group was occur during the study.
deemed necessary in this research because clinical reference Hematology and serum mineral analysis. Whole-blood mea-
ranges for hematology and serum biochemistry values are surements consisted of hemoglobin, total white cell count,
not currently well-established for African children.52 Thus, and platelet count. Whole-blood analysis was conducted with
UPSN INTERVENTION IN CHILDREN 779
a flow cytometer (Abx micro60; Block Scientific, Bohemia, trend in the calculated body mass index (BMI) values for
NY). Serum albumin, alkaline phosphatase (ALP), alanine participants versus the percentage reduction of AFM1 levels
aminotransferase (ALT), aspartate aminotransferase (AST), in urine. Although the average height-for-age z score was
gamma-glutamyl transpeptidase (GGT), total protein, total −1.2 ± 1.8, values ranged from −6.01 to 2.14, indicating that
bilirubin, urea, creatinine, triglycerides, sodium (Na+), potas- nutritional status varied significantly in the population.
sium (K+), chloride (Cl−), calcium (Ca2+), and magnesium Adherence to the 2-week study protocol was excellent, with
(Mg2+) were measured using a Flexor E automatic blood all 63 participants completing the study. Only one participant
analyzer (Vital Scientific, Dieren, Netherlands). missed an evening dose of treatment throughout the 28 doses
AFM1 analysis. Analysis of urinary AFM1 levels followed per participant administered. This participant was in the low-
previously published methods38,53 that have been validated dose UPSN group and diagnosed with and treated for malaria
by our laboratory.44 After immunoaffinity column (Aflatest that same day. General acceptance by the parents and chil-
WB; VICAM) clean up, urine samples were analyzed using dren was exceptional.
a high-pressure liquid chromatography (HPLC) system with Adverse events and side effects. The two dose levels of
fluorescence detection capability (Shimadzu Corp., Kyoto, UPSN (0.75 and 1.5 g/day) were tolerable to the participants
Japan). Urinary AFM1 concentrations were expressed as throughout the study. Adverse symptoms reported during
picograms per milligram creatinine to correct for variations the 2-week study were primarily of a gastrointestinal nature
in urine dilution among samples. Creatinine concentrations and included vomiting and diarrhea. Table 2 is a tally of
were measured by a Flexor E Autoanalyzer. individual adverse events reported throughout the course of
Statistical analysis. Statistical analysis was conducted with the study. It is important to note that, in some cases, reports
JMP 10 software (SAS Institute, NC). The ultimate goal of this were made multiple times by the same participant. For exam-
study was to determine if the ingestion of UPSN clay was ple, all reported events in the high-dose group originated
safe in children; therefore, statistical evaluation focused on from one individual who received medical attention from
comparisons between treatment arms as well as values within the district hospital after 2 consecutive days of vomiting. This
a group at baseline and day 15. A c2 test was used for analysis participant was diagnosed and treated for malaria after visit-
of side effect/toxicity data between treatment groups. As ing the hospital but allowed to stay in the clinical trial per the
expected, the AFM1 biomarker of exposure data was not nor- physician’s recommendation. Vomiting ceased after initiation
mally distributed and therefore, was log-transformed before of malarial medication. In total, there were three children
analysis. Paired t test and analysis of variance (ANOVA) sta- diagnosed and treated for malaria during the study. This is
tistical tests were conducted on both AFM1 data and bio- equivalent to 4.8% of the study population, which is actually
chemical parameters for comparisons among treatment groups. a lower prevalence rate than reported for the area (> 75 per
A two-tailed P value < 0.05 was considered statistically signifi- 1,000) by the WHO.54 The symptoms reported by indi-
cant. Correlation analyses were performed for serum biochemi- viduals with malaria accounted for 9 of the total 13 adverse
cal parameters and AFM1 levels. P values and correlation events (69.2%) reported. The placebo, low-dose, and high-
coefficients were calculated by a Pearson correlation test to dose groups experienced side effects at rates of 0.3% (2 of
evaluate the association between bilirubin and AFM1 levels. 588), 0.8% (5 of 588), and 1% (6 of 588), respectively.
Severities of side effects were generally reported as mild
to moderate, and either no treatment or self-treatment was
RESULTS
effective in alleviating symptoms. Severe cases of vomiting
Study participant characteristics and compliance. In total, requiring immediate medical attention occurred only in those
63 child participants were enrolled in the clinical trial. There
were no significant differences in mean age, sex, weight, or Table 2
other general physical parameters, such as blood pressure, Adverse events reported
between treatment groups (Table 1). Also, there was no Treatment group
Placebo Low dose High dose Sum
Table 1 Symptom reported
Demographics and physical parameters Other 0* 0 2† 2
Treatment group Indigestion 0 0 0 0
Nausea 0 0 0 0
Demographic characteristics Placebo Low dose High dose
Vomiting 2 3† 4† 9
Participants 21 21 21 Constipation 0 0 0 0
Sex Diarrhea 0 1 0 1
Male 13 14 14 Flatulence 0 0 0 0
Female 8 7 7 Loss of appetite 0 1 0 1
Age (years)* 5.8 ± 1.6 5.4 ± 1.8 5.7 ± 1.9 Abdominal discomfort 0 0 0 0
Body weight (kg)* 20.0 ± 4.8 21.0 ± 8.5 18.9 ± 5.2 Heartburn 0 0 0 0
Height (cm)* 107.2 ± 13.2 107.9 ± 16.3 105.8 ± 15.1 Dizziness 0 0 0 0
Height-for-age z score −1.5 ± 1.9 −0.8 ± 1.8 −1.4 ± 1.6 Insomnia 0 0 0 0
Weight-for-age z score −0.4 ± 1.5 0.2 ± 1.0 −0.6 ± 1.2 Bloating 0 0 0 0
BMI (kg/m2) 17.3 ± 1.8 17.7 ± 3.3 16.7 ± 1.7 Total incidence (%) 2 (0.3) 5 (0.8) 6 (1) 13 (0.7)
Systolic blood 87.7 ± 8.0 91.3 ± 10.9 92.3 ± 11.0 Severity
pressure (mmHg)* Mild 2 2 5 9
Diastolic blood 44.6 ± 7.3 45.6 ± 9.1 47.2 ± 10.2 Moderate 0 2 1 3
pressure (mmHg)* Severe 0 1 0 1
Note that all data are baseline values. *Indicates number of times an adverse event was reported.
*Mean ± SD. †Indicates that the participant was diagnosed with malaria by health officials.
780 MITCHELL AND OTHERS
Table 3
Hematological analysis
Treatment group
Placebo Low dose High dose
Normal clinical range
Before After Before After Before After (United States) Reference
Hemoglobin (g/dL) 11.3 ± 0.8 11.4 ± 1.1 11.5 ± 0.9 11.7 ± 1.3 11.0 ± 1.5 11.3 ± 1.5 11.0–14.5 A
WBC ( +1,000/mm3) 8.2 ± 2.0 7.4 ± 1.2 7.8 ± 2.0 7.9 ± 1.8 8.5 ± 3.4 8.2 ± 2.7 3.4–12.0 A
Platelet ( +1,000/mm3) 275.3 ± 73.9 303.0 ± 112.4 296.0 ± 85.0 349.9 ± 142.7 266.0 ± 80.4 280.9 ± 117.3 150.0–450.0 A
Lymphocytes (%) 55.2 ± 10.9 56.9 ± 8.1 52.6 ± 7.9 51.8 ± 9.6 52.9 ± 11.2 53.5 ± 9.4 28.0–48.0 B
Monocytes (%) 10.6 ± 3.2 11.0 ± 3.9 12.3 ± 3.7 13.0 ± 3.9 10.4 ± 3.0 10.9 ± 3.3 3.0–6.0 B
Granulocytes (%) 34.2 ± 12.1 31.9 ± 7.4 35.1 ± 8.4 35.2 ± 10.7 36.6 ± 11.0 35.6 ± 9.1 32.0–76.0 B
Data are mean ± SD. A indicates Mayo Clinic pediatric reference values. B indicates Children’s Hospitals and Clinics of Minnesota reference values. Reference ranges are combined for males
and females. WBC = white blood cell.
participants later diagnosed with malaria. Importantly, there age (data not shown). Although treatment groups were not
were no significant differences observed in the numbers of significantly different at baseline or day 15 of treatment, the
adverse events between treatment groups (P = 0.37) or the high-dose UPSN group exhibited significantly (P = 0.0216)
severity of symptoms reported (P = 0.43). lower AFM1 levels on day 7 than both the placebo and low-
Hematological, blood chemistry, and serum mineral effects. dose groups. When all data values from days 7 and 15 were
Hematological analysis of blood samples indicated no signifi- pooled by treatment group, the high-dose group was still sta-
cant difference between treatment arms, although lymphocytes tistically lower (P = 0.0063) and showed a 52% reduction
and monocytes were above the normal range across all treat- in median AFM1 levels compared with control (Figure 2).
ment groups at both days 0 and 15 (Table 3). Hematological Pooled AFM1 levels for placebo, low-dose, and high-dose
values in the placebo group were not significantly different groups were 549.00 (95% confidence interval [95% CI] =
from either the low- or high-dose UPSN groups at day 0 or 15. 416.58–681.42), 171.33 (95% CI = 150.46–192.20), and 121.87
These levels were also not significantly different within each (95% CI = 103.01–140.73), respectively.
treatment group comparing day 15 values with baseline values.
Results of serum biochemistry analyses are provided in DISCUSSION
Table 4. No significant differences were observed within groups
between days 0 and 15 for albumin, ALP, AST, GGT, total Chronic childhood AF exposure has gained interest over
bilirubin, urea, or triglycerides. ALT values were significantly the past decade as a potential variable in the complex milieu
higher after treatment in all groups (P < 0.0003), including the of biological and environmental factors that lead to stunting,
placebo group. The placebo group also exhibited significantly wasting, and suppressed immunity. In particular, sub-Saharan
lower (P < 0.01) total protein levels after treatment; how- Africa has been identified as an area at high risk for AF
ever, this was not the case for the UPSN-treated groups. exposure as well as growth stunting. Investigations from the
At day 15, the low-dose UPSN group had significantly lower Ejura district in the Ashanti Region of Ghana have shown
creatinine levels compared with baseline (P < 0.05); how- ongoing, high-level AF exposure over the past decade.44,45,55,56
ever, this observation was not dose-dependent. Comparison A prevalence of up to 54.9% has been reported for stunted
of serum biochemistry parameters between the placebo group and/or wasted children from another district of the Ashanti
and the low- or high-dose UPSN group did not indicate sig- Region.57 Although the high rates observed in this population
nificant differences at day 0 or day 15 (Table 4). Although are primarily thought to occur as a result of inadequate nutri-
average total bilirubin levels were within range in all treat- tion and protein intake, multiple variables likely contribute
ment groups throughout the study, a wide range of values was to the etiology of disease. Chronic AF exposure in this com-
detected (0.2–35.5 mmol/L), and a weakly positive but signifi- munity could be one such contributing factor, particularly
cant (P = 0.005) association with urinary AFM1 concentration after administration of nutritional supplements such as home-
at day 0 was noted (Figure 1). made Weanimix, which consists of groundnuts, beans, and
Median, mean, and normal US pediatric ranges for the maize (0.5:0.5:4 ratio). A recent assessment of the weaning
serum minerals assessed are outlined in Table 5. Serum Na+ foods produced in this community intended for children
and Cl− significantly increased (P < 0.01) over the course of between the ages of 6 months and 2 years showed AF
the study in all groups. All groups exhibited significantly contamination in 100% of samples, with levels as high as
decreased Ca+ levels after the study. A reduction in serum 500 ppb.58 Urine samples collected from children before and
Mg2+ was seen in the UPSN high-dose group (P < 0.01). There after 21 days of homemade Weanimix consumption revealed
was also a small reduction in the mean Mg2+ of the UPSN increased levels of urinary AFM1, indicating that, although
low-dose group; that was not significantly different (P = it is an important nutritional supplement in this region,
0.27). However, levels in all groups remained well within Weanimix can also cause heightened AF exposure.58 There-
the normal US range. No other mineral level comparison fore, an intervention strategy to reduce childhood exposure
between days 0 and 15 was significantly different. in these populations while maintaining the use of these impor-
AFM1 biomarker levels in urine. Throughout the study, tant nutritional supplements is of particular interest.
100% of urine samples contained detectable levels of AFM1. Enterosorption therapy may be a valuable tool in low-
AFM1 concentration ranged from 0.5 to 5443.7 pg/mg creati- economic, high-risk areas, where food insecurity results in
nine. The mean at day 0 was 297.8 pg/mg creatinine. AFM1 limited variety in the diet and continued consumption of poor-
levels were not significantly different by sex or correlated with quality foods.37,59 Clinical trials using similar dioctahedral
UPSN INTERVENTION IN CHILDREN 781
Table 4
Serum biochemistry
Treatment groups
Placebo Low dose High dose
Normal clinical range
Before After Before After Before After (United States) Reference
Albumin (g/L)
49 47.2 48.5 47.9 49.6 48.4
47.0 ± 9.0 46.9 ± 2.8 48.5 ± 3.1 47.2 ± 2.3 46.2 ± 11.1 47.6 ± 6.0 35.0–50.0 A
15.9–56.0 39.7–51.0 42.5–53.0 42.2–51.6 11.1–57.4 25.2–56.2
ALP (U/L)
591.9 596.2 479 620.5 521.3 581.9
559.1 ± 185.7 532.0 ± 355.6 427.2 ± 242.3 572.3 ± 332.6 488.5 ± 215.8 584.9 ± 314.7 149.0–468.0 A
176.9–1,039.4 6.3–1,164.3 17.3–843.4 6.1–1,102.2 13.1–907.6 24.7–1,189.1
ALT (U/L)
13.2 27.1 17.2 27.6 17.7 24.6
14.4 ± 6.0 430.2 ± 11.3* 19.3 ± 8.6 28.9 ± 7.2* 20.2 ± 20.0 29.3 ± 15.1* 7.0–55.0 A
4.3–27.1 13.7–51.4 7.5–46.5 20.8–45.8 3.0–87.3 12.4–68.3
AST (U/L)
39.8 41.2 38.3 42.1 38.4 40.4
41.8 ± 13.2 44.9 ± 12.3 42.3 ± 8.4 43.7 ± 10.8 40.2 ± 14.1 41.4 ± 10.0 8.0–60.0 A
16.8–66.3 31.4–73.3 30.9–59.5 29.8–74.3 12.6–85.9 20.6–57.3
GGT (U/L)
15 16.6 10.6 14 13.2 15
16.6 ± 8.0 17.0 ± 6.8 13.9 ± 8.6 16.0 ± 7.3 14.1 ± 9.0 17.0 ± 11.7 7.0–29.0 A
1.7–42.0 6.1–31.4 6.4–43.2 7.4–35.5 0.5–37.2 2.8–48.7
Total bilirubin (mmol/L)
5.5 8.1 6.2 7.5 6.7 6.1
7.1 ± 7.1 8.5 ± 4.9 7.8 ± 5.4 7.9 ± 3.4 8.4 ± 8.1 8.3 ± 7.4 1.7–17.1 A
0.2–28.1 0.6–22.4 0.5–25.4 1.8–15.9 1.1–35.5 0.2–29.9
Total protein (g/L)
80.5 76.3 77.8 77.2 79.8 80.9
78.8 ± 8.8 77.0 ± 3.3* 78.2 ± 6.0 77.4 ± 4.3 79.8 ± 31.0 78.0 ± 8.6 63.0–79.0 A
43.6–87.2 70.8–82.5 70.2–89.8 70.1–86.1 21.3–194.2 47.8–89.6
Urea (mmol/L)
2.9 2.7 2.8 2.7 3.1 2.8
2.7 ± 0.8 2.9 ± 0.6 2.8 ± 0.8 2.6 ± 0.6 2.9 ± 1.2 2.9 ± 1.0 2.5–7.1 A
0.8–3.8 2.1–4.5 1.5–4.6 1.6–3.7 0.6–5.2 1.6–6.0
Creatinine (mmol/L)
58.1 53.3 61.7 54.3 64.4 59.1
54.6 ± 16.4 54.3 ± 6.2 62.4 ± 10.1 55.7 ± 9.5* 60.7 ± 17.9 56.5 ± 9.0 50.0–110.0 C
5.4–71.1 40.2–65.1 38.7–77.0 42.4–78.5 18.5–90.4 34.1–74.2
Triglycerides (mmol/L)
0.8 1.1 1 1 0.9 1.1
1.0 ± 0.7 1.3 ± 0.8 1.3 ± 0.7 1.2 ± 0.7 1.6 ± 3.0 1.3 ± 0.6 < 1.02 A
0.2–2.6 0.4–3.5 0.5–3.0 0.7–3.8 0.2–14.1 0.5–3.2
Data represent median, mean ± SD, and range. A indicates Mayo Clinic pediatric reference ranges. C indicates Royal College of Physicians and Surgeons of Canada ranges. Reference
ranges are combined male and female values.
*Denotes significant difference between baseline and after treatment.
smectite clays at doses as high as 6 g/day for the treatment placebo group experienced the greatest increase in ALT,
of acute diarrhea in children resulted in limited adverse although day 15 levels were still well within the normal
effects, of which mild constipation was the most severe range for all three groups. Sodium and chloride ion levels
event reported.60–63 Similarly, results from this study indi- were also significantly increased over the study period;
cate that administration of dietary UPSN powder at con- however, this was observed in both placebo and UPSN-
centrations from 0.75 to 1.5 g/day in healthy children (ages treated groups, indicating little biological significance. The
3–9 years) for 14 days resulted in minimal side effects. levels for Cl− were still within the normal reference range,
Neither dose-dependent toxic effects nor severe clinical whereas some Na+ values were out of range based on the
symptoms were related to UPSN consumption in this study. US pediatric values, which is shown in Table 5.65
Hematologic parameters indicated that UPSN treatment did Increases in Ca2+ have been observed after administra-
not impair immunity or promote an inflammatory response.64 tion of the parent NS product and UPSN in rats, which was
ALT values were increased in all treatment groups at day attributed to dissolution of calcite and exchangeable Ca2+
15; however, there were no significant differences between ions from montmorillonite.46,47 However, in this study, total
the low- and high-dose UPSN groups and the placebo group. Ca2+ levels were decreased in all treatment groups, including
These values were also within normal pediatric reference the placebo (calcium carbonate). Calcium carbonate and, to
ranges reported by the US Mayo Clinic (Table 4). Addi- a lesser extent, NS clay typically act as calcium supplements;
tionally, all other liver toxicity parameters (i.e., ALP, AST, thus, it is likely that this overall reduction in serum levels is
bilirubin, and GGT) were not increased at day 15 in any a result of dietary changes during the intervention trial.
treatment groups. Therefore, the cause of increased ALT Magnesium ion was the only serum micronutrient that
values over the duration of the study remains unclear. The dose-dependently decreased with UPSN treatment. The mean
782 MITCHELL AND OTHERS
Figure 1. Correlation of log-transformed total bilirubin and Figure 2. Comparison of urinary AFM1 levels for placebo and
log-transformed AFM1 values for all participants at day 0. low- and high-dose treatment groups. AFM1 data for each group were
pooled for days 7 and 15. *Indicates statistical significance (P < 0.05).
concentrations in the high-dose group were significantly lower
than in the placebo group on day 15 after treatment, and the Longer safety trials controlling for intake of essential dietary
low-dose group, although not significantly different, showed nutrients are warranted to determine whether UPSN could
a decreasing trend from the placebo. However, it is important interfere with micronutrient or mineral absorption in children.
to note that all levels remained within the normal range Although changes in serum bilirubin have been reported
throughout the study. Furthermore, significant modulation in after exposure to AF in animal species, to our knowledge,
serum Mg2+ concentrations has not been observed in any other there have been no correlations made between AF exposure
animal or human study with UPSN or parent NS. However, and bilirubin levels in humans.68,69 The AFB1-albumin
decreased absorption and retention of Mg2+ were observed adduct, although a valuable AF assessment tool in the serum,
in a pig model after ingestion of 1% sodium montmorillonite is a long-term biomarker of exposure and not known to fluc-
clay.66 Magnesium levels are controlled by the kidneys and tuate with recent exposure as rapidly as the urinary bio-
gastrointestinal tract and seem to be closely linked to calcium, marker. For this reason, urinary AFM1 is a more appropriate
potassium, and sodium metabolism.67 Therefore, the change marker to correlate with dynamic serum components, such as
in Mg2+ observed here could have resulted from changes in bilirubin. As stated previously, AF has been shown to elevate
calcium or sodium metabolism and not directly from UPSN total bilirubin and ALP levels in animal models. Because
treatment. An alternative explanation for the lowered serum growth stunting has been reported to be common in most
Mg2+ observed is a direct sequestration of Mg2+ by UPSN forms of chronic liver disease, it will be important to assess
in the gut through cation exchange activity of the clay, thus liver function parameters in future studies involving children,
reducing the availability of Mg2+ for absorption from the gut. growth stunting, and AFs.70 Additionally, because direct
Table 5
Serum mineral analysis
Treatment groups
Placebo Low dose High dose
Normal clinical range
Before After Before After Before After (United States) Reference
Na (mmol/L)
132.2 138.2 135.3 138.1 128.5 135.6
130.6 ± 6.8 139.2 ± 5.5* 133.6 ± 8.0 138.8 ± 3.8* 129.3 ± 9.1 136.8 ± 3.1* 135.0–145.0 A
115.3–141.4 132.2–153.4 114.3–146.2 134.5–150.3 112.1–149.9 132.7–143.3
K (mmol/L)
3.7 4 3.8 3.9 3.7 4.1
3.8 ± 0.4 4.0 ± 0.4 3.8 ± 0.4 4.1 ± 0.5 3.8 ± 0.5 4.1 ± 0.4 3.6–5.2 A
3.0–4.8 3.3–5.0 2.7–4.5 3.2–5.7 3.1–4.8 3.3–4.7
Cl (mmol/L)
97.3 101.2 98.8 102.4 96.2 100.4
97.4 ± 4.2 101.2 ± 4.0* 97.7 ± 5.6 101.5 ± 2.5* 96.6 ± 5.1 100.3 ± 2.1* 102.0–112.0 A
89.4–105.3 90.1–108.4 83.0–106.8 96.0–104.8 87.8–106.3 95.4–105.2
Ca (mmol/L)
2.4 2.1 2.3 2.2 2.3 2.1
2.4 ± 0.2 2.2 ± 0.3* 2.3 ± 0.1 2.2 ± 0.2* 2.3 ± 0.2 2.1 ± 0.3* 2.4–2.7 A
1.9–2.7 1.8–2.8 2.0–2.6 1.2–2.6 2.0–2.6 1.5–2.5
Mg (mmol/L)
0.9 0.8 0.9 0.8 0.8 0.8
0.8 ± 0.1 0.8 ± 0.1 0.9 ± 0.1 0.8 ± 0.1 0.8 ± 0.1 0.8 ± 0.1* 0.7–1.0 A
0.5–1.0 0.6–1.0 0.7–1.0 0.6–1.0 0.7–1.1 0.6–0.9
Data represent median, mean ± SD, and range. A indicates Mayo Clinic pediatric reference ranges. Reference ranges are combined male and female values.
*Denotes statistical significance between baseline and after treatment.
UPSN INTERVENTION IN CHILDREN 783
bilirubin can be measured in the urine of individuals expe- 5. Gong YY, Egal S, Hounsa A, Turner PC, Hall AJ, Cardwell KF,
riencing liver malfunction, it may be an excellent non-invasive Wild CP, 2003. Determinants of aflatoxin exposure in young
children from Benin and Togo, West Africa: the critical role
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Although the range of AFM1 excretion was similar, the 6. Turner PC, Collinson AC, Cheung YB, Gong Y, Hall AJ,
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those seen in adults from the same population in October causes growth faltering in Gambian infants. Int J Epidemiol
of 2010.45 This finding may be explained by the fact that 36: 1119–1125.
7. Turner PC, Moore SE, Hall AJ, Prentice AM, Wild CP, 2003.
this intervention trial was carried out during the wet season, Modification of immune function through exposure to dietary
whereas the adult study took place at the beginning of the aflatoxin in Gambian children. Environ Health Perspect 111:
dry season, which typically correlates with increasing AF 217–220.
exposure.16,71 Also, the variance in excretion levels could 8. Shuaib FM, Jolly PE, Ehiri JE, Yatich N, Jiang Y, Funkhouser
be attributed to the difference in food intake, metabolism, E, Person SD, Wilson C, Ellis WO, Wang JS, Williams JH,
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and urinary output between children and adults. The high- biomarker blood levels in pregnant women in Kumasi, Ghana.
dose UPSN group showed a significant decrease in AFM1 Trop Med Int Health 15: 160–167.
excretion compared with the placebo group (52%). This 9. Abdulrazzaq YM, Osman N, Yousif ZM, Trad O, 2004. Mor-
decrease in AFM is similar to the percentages previously bidity in neonates of mothers who have ingested aflatoxins.1
reported after NS and UPSN consumption in adults (45%, Ann Trop Paediatr 24: 145–151.
55%, and 58.7%).44,45
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that UPSN consumption by children (ages 3–9 years) is safe 11. Bhutta ZA, Ahmed T, Black RE, Cousens S, Dewey K, Giugliani
at a dose up to 1.5 g/day for 2 weeks. Inclusion of UPSN in E, Haider BA, Kirkwood B, Morris SS, Sachdev HP, Shekar
M, 2008. What works? Interventions for maternal and child
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adverse effects of AF exposure and enhancing the quality, effi- vention of childhood undernutrition. Nutr Rev 68: 429–435.
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Received February 10, 2014. Accepted for publication June 24, 2014.
nutrient status of Ghanaian infants from 6 to 12 mo of age.
Published online August 18, 2014. Am J Clin Nutr 70: 391–404.
14. Kumi J, Nicole M, Asare GA, Dotse E, Kwaa F, Phillips T,
Acknowledgments: The authors would like to acknowledge the sup- Ankrah N-A, 2013. Aflatoxins and fumonisins contamination
port and work put forth by the study monitors and health officials of home-made food (weanimix) from cereal-legume blends for
at the Ejura District Hospital in the Ashanti Region of Ghana. children in Ghana. Toxicol Lett 221: S119
Financial support: This work was funded by US Agency for Inter- 15. Polychronaki N, Wild C, Mykkänen H, Amra H, Abdel-Wahhab
national Development—Peanut Collaborative Research Support Pro- M, Sylla A, Diallo M, El-Nezami H, Turner P, 2008. Urinary
gram Grant USAID LAG-G-00-96-90013-00. biomarkers of aflatoxin exposure in young children from Egypt
and Guinea. Food Chem Toxicol 46: 519–526.
Disclaimer: The authors do not have any conflicts of interest with 16. Turner PC, Mendy M, Whittle H, Fortuin M, Hall AJ, Wild CP,
regard to the research reported in this manuscript. 2000. Hepatitis B infection and aflatoxin biomarker levels in
Authors’ addresses: Nicole J. Mitchell, Department of Food Science Gambian children. Trop Med Int Health 5: 837–841.
and Human Nutrition, Michigan State University, East Lansing, MI, 17. Turner PC, Sylla A, Kuang SY, Marchant CL, Diallo MS,
E-mail: mitch441@anr.msu.edu. Justice Kumi and Nii-Ayi Ankrah, Hall AJ, Groopman JD, Wild CP, 2005. Absence of TP53
Noguchi Memorial Institute for Medical Research, University of codon 249 mutations in young Guinean children with high
Ghana, Legon, Accra, Ghana, E-mails: JKumi@noguchi.ug.edu.gh aflatoxin exposure. Cancer Epidemiol Biomarkers Prev 14:
and NAnkrah@noguchi.ug.edu.gh. Mildred Aleser, Ejura-Sekyedumase 2053–2055.
District Hospital, Ejura, Ghana, E-mail: msgbireh@yahoo.com. Sarah 18. Wild C, Fortuin M, Donato F, Whittle H, Hall A, Wolf C,
E. Elmore, Kristal A. Rychlik, Katherine E. Zychowski, Amelia A. Montesano R, 1993. Aflatoxin, liver enzymes, and hepatitis B
Romoser, and Timothy D. Phillips, Veterinary Integrative Biosciences, virus infection in Gambian children. Cancer Epidemiol Bio-
College of Veterinary Medicine and Biomedical Sciences, Texas A&M markers Prev 2: 555–561.
University, College Station, TX, E-mails: selmore@cvm.tamu.edu, 19. Wild C, Jiang Y, Allen S, Jansen L, Hall A, Montesano R, 1990.
kabrown@cvm.tamu.edu, kzychowski@cvm.tamu.edu, aromoser@cvm Aflatoxin-albumin adducts in human sera from different regions
.tamu.edu, and tphillips@cvm.tamu.edu. of the world. Carcinogenesis 11: 2271–2274.
20. Obuseh FA, Jolly PE, Jiang Y, Shuaib FM, Waterbor J, Ellis
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