Assessment of Nutrient Profile, Nutritional Status, Development and Severity of Children With Autistic Spectrum Disorder in Comparison With Typically Developing Children in Addis Ababa, Ethiopia
Conditions: Autistic Spectrum Disorder
Sponsor: Addis Ababa University

 
ASSESSMENT OF NUTRIENT 
PROFILE, NUTRITIONAL STATUS, 
DEVELOPMENT AND SEVERITY 
OF CHILDREN WITH AUTISTIC 
SPECTRUM DISORDER IN 
COMPARISON WITH TYPICALLY 
DEVELOPING CHILDREN IN 
ADDIS ABABA, ETHIOPIA 
August 19, 2022 
 
 
 

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Table of contents 
Study Protocol with SAP ......................................................................................... 1 
1. Scope ................................................................................................................... 1 
2 DEFINITION OF TERMS................................................................................... 1 
2. Objectives ........................................................................................................... 2 
2.1. 
General objectives ............................................................................................................ 2 
2.2. 
Specific objectives............................................................................................................ 2 
3. Materials ............................................................................................................. 2 
4. Subjects ............................................................................................................... 3 
4.1. 
Inclusion criteria ............................................................................................................... 3 
4.2. 
Exclusion criteria.............................................................................................................. 3 
4.3. 
Test conditions ................................................................................................................. 3 
5. Procedure ............................................................................................................ 4 
5.1. 
Height ............................................................................................................................... 4 
5.2. 
MUAC (Middle Upper Arm Circumference)................................................................... 4 
5.3. 
Weight .............................................................................................................................. 4 
5.4. 
Urine collection ................................................................................................................ 5 
5.5. 
CARS diagnosis ............................................................................................................... 5 
5.6. 
Food sample collection..................................................................................................... 5 
6. Analysis ............................................................................................................... 5 
6.1. 
Proximate analysis............................................................................................................ 5 
6.2. 
Urine iodine analysis ........................................................................................................ 7 
6.3. 
Statistical analysis plan .................................................................................................... 7 

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STUDY PROTOCOL WITH SAP 
1. SCOPE 
This study protocol specifies objective, design and methods for the assessment of assessment of 
nutrient profile, nutritional status, development and severity of children with autistic 
spectrum disorder in comparison with typically developing children in Addis Ababa, 
Ethiopia  
2 DEFINITION OF TERMS   
 
a) AL-cup: the type of glass and non-glass cups used exclusively for laboratory purposes. 
For instance in the analysis of fat.  
b) Desiccator: a glass container or other apparatus holding a drying agent for removing 
moisture from sample and protecting them from surrounding moisture.  
c) Digestion: heating of a sample in extreme temperatures using various reagents that 
corresponds to the type of analysis.  
d) PH: measure of level of acidity and Alkalinity (base) on a scale of 1 to 14. Lower PH 
indicating higher acidity while higher PH indicates the alkalinity of a substance.  
e) Cruisable: ceramic bowl container used in the analysis of samples that involve direct 
heat exposer or of stored in an oven with elevated temperature      
f) Titration: a slow addition of a solution of a known concentration (titrant) to a known 
solution of unknown concentration until the reaction reaches neutralizations, which is 
often indicated by a change of color.  
g) Muffle furnace: a type of jacketed enclosure that is used to heat a material to a 
significantly high temperature while keeping it contained and fully isolated from external 
contaminants.  
h) Spectrophotometry: a method of measuring how much a chemical substance absorbs 
light by measuring the intensity of light as a beam of light passes through sample 
solution.  

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i) Colorimetery: a method of determining the concentration of a chemical agent with the 
aid of a color reagent and its light absorption.  
j) Thimble:  apparatus of fat analysis equipment (soxhlet extractor) that is usually made of 
thick filter paper which retains the solid to be extracted.            
2. OBJECTIVES  
2.1. General objectives 
 
The general objective of this study is to assess the nutritional status, nutrient profile, ASD 
severity, and development of children diagnosed with ASD as compared to typically developing 
controls in Addis Ababa, Ethiopia. 
2.2. Specific objectives  
 
The specific objectives of this study are to:- 
 
1. Evaluate the nutrient profile of diets using proximate analysis.  
2. Determine the nutritional status (i.e. stunting, wasting, underweight and overweight) of 
children diagnosed using anthropometric measurements. 
3. Assess the dietary diversity score (DDS) of children using food frequency questioners (FFQ). 
4. Examine the urine samples of children to identify the urine iodine (UI) level. 
5. Assess the parents and caregivers perceptions about the Eating behaviour of ASD children 
using questioner. 
6. Evaluate the ASD severity level and developmental growth of children with ASD in 
correlation to UI level. 
   
3. MATERIALS 
a) Weight scale 
b) Height board measurement 
c) Middle Upper Arm Circumference tape  
d) CARS standard version rating booklet 

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e) Food collection Bag 
f) Urine collection cup 
g) Proximate and urine analysis equipments and chemicals ( hexane, H2SO4, Ethanol, 
digester, soxhlet extractor, etc.)  
4. SUBJECTS 
Selection of 26 children with Autistic Spectrum Disorder (ASD) (case group) and the 
corresponding 26 typically developing children (control group), 52 children in total, shall be 
made on the basis of:  
4.1.Inclusion criteria 
a) Children that are already diagnosed with ASD for the “case” group,  
b) Children fulfilling the matching criteria to the case group (i.e. age and sex) for the 
control group.  
4.2.Exclusion criteria 
a) Use of special supplements,  
b) Use of heavy medication,  
c) Children with critical chronic disease such as cancer, HIV AIDs.  
4.3.Test conditions 
The subjects shall: 
a) Be of school age;  
b) Legal guardians of the child need to Sign a consent form for the willingness of his/her 
participation; 
c) Give the necessary information and specimen agreed upon in the given consent forms.  
The analysis shall: 
a) Be conducted by  

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 The researcher as per guidelines and procedures described for all nutritional 
analysis.  
 Registered nurse for urine sample collection  
 Registered physician for CARS diagnosis.    
b) Have no risk involved since it doesn’t involve any penetration to the human muscle    
c) Be done with the willingness of the participant and their legal guardians (he/she can stop 
the participation anytime) 
5. PROCEDURE 
5.1. Height 
A child height board will be used to measure height of the children. The children will be 
measured with minimal clothing and no shoes and socks must be worn. The feet must be 
together, arms to the side, legs straight and shoulders relaxed, the head must be in the Frankfort 
horizontal plane (looking straight ahead). An average of 3 readings will be taken and read to the 
nearest 0.1 cm. 
 
5.2. MUAC (Middle Upper Arm Circumference)  
MUAC is the circumference of the left upper arm and is measured at the mid-point between the 
tips of the shoulder and elbow using a MUAC tape. To measure: 
Bend the left arm, find and mark with a pen the olecranon process and acromium. 
 Mark the mid-point between these two marks. 
 With the arm hanging straight down, wrap a MUAC tape around the arm at the midpoint 
mark. 
 Measure to the nearest 1 mm 
 
5.3. Weight 
The children will be measured using an electronic weight scale with minimal clothing and no 
shoes and socks must be worn. The scale will be placed on a hard, flat (level) surface up on 
measurement and scale reading 

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5.4. Urine collection 
60 ml. urine samples will be collected from each child by a trained clinical practitioner (nurses), 
at the child’s period of urinary stimulation, using sample collection cups.  
 
5.5. CARS diagnosis  
CARS is scale that is frequently used in the diagnosis of ASD in children. It consists of 15 items 
that cover different symptoms of ASD, and provides a reliable comparison of an affected child’s 
behaviors and skills against the expected developmental growth of a healthy child. The diagnosis 
will be conducted by a physician by combining parent reports (direct question and answer) and 
direct observations of children made by the clinician.    
 
5.6.  Food sample collection 
50gm sample of food will be collected in sample collection bags from each meal consumed 
throughout the day by the researcher and legal guardians.    
6. ANALYSIS  
6.1. Proximate analysis    
A) Moisture content: An Al-cup will be dried at 104°Cfor 30 minutes and cooled in a 
desiccator, and record the weight (W1). 3 g of the sample will then be added in the Al-cup 
and weighed (W2). The sample will then be dried for two hours at 139 °C. Then, the final 
dried sample will be weighed (W3) and recorded after cooling in a desiccator. The net 
moisture content of the sample will then be calculated using the following formula. %MC= 
((W2-W1)-W3) ×100/ W1-W3 (%MC = percentage of net moisture content, W1= weight of 
Al-cup, W2= weight of sample before drying, W3= weight of sample after drying. 
B) Crude protein: The amount of protein present is calculated using the Kjeldahl method of 
protein analysis. The food sample will be weighed (0.5-1g) into a digestion flask. The sample 
will then be digested by heating it in the presence of sulfuric acid, a catalyst, copper (0.5g), 
and potassium sulfate (100g). Digestion will convert any nitrogen in the food into ammonia, 
and other organic matter to C02 and H20. After the digestion has been completed the 

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digestion flask will be connected to a receiving flask by a tube. The solution in the digestion 
flask will be made alkaline by adding sodium hydroxide, which converts the ammonium 
sulfate into ammonia gas. The ammonia gas that is formed will be liberated and mixed with 
boric acid. The low pH of the solution will convert the ammonia gas into the ammonium ion, 
and will simultaneously convert the boric acid to the borate ion. The nitrogen content will 
then be estimated by titration of the ammonium borate formed with standard hydrochloric 
acid using a suitable indicator to determine the end-point. The following equations will be 
used to determine the nitrogen concentration of a sample and corresponding crude protein 
percentage. %N = {(Vs - Vb) *molecular weight of N * N Hcl}/sample weight*10, % protein 
= 6.25* %N (Vs= sample volume, Vb= blank volume, N Hcl = normality of Hydrochloric 
acid.)  
C) Ash content: A cruisable will first be weighed on electronic balance (W1). Afterward, 3g of 
food sample will be measured using an electronic balance (W2). The samples will then be put 
on the hot plate and charred and will be placed in a muffle furnace for 2 hours at 550°C. The 
final weight will then be measured after cooling it down (W3). The ash content will then be 
calculated using the formula: Ash % = W3 – W1 × 100 W2 
D) Fat content (AOAC, 2000): Al-cup will first be heated for 30 minutes at 106°C, cooled on a 
desiccator, and weighed on electronic balance (W1). 1-5g of food sample will then be added 
on a thimble and weighed (W2). The thimble and Al-cup will be slot in the corresponding 
positions of the fat extraction unit. Subsequently, 70% of diethyl ether will be added to the 
sample in the thimble using a dispenser. The hot plate temperature will then be set at 
500°Cfor 20 minutes for soaking followed by the extraction step for 40 minutes. The third 
step will be the evaporation step. After the extraction step is complete, the extracted fat will 
be placed in an oven for 30 minutes at 106°C. The fat content will then be determined using 
the following formula (W3) % of fat= W3-W1 ×100 W2 
E) Total dietary fiber: duplicate portions will be incubated with pancreatic α- amylase and 
amyloglycosidase for 16 hr. at 370°C in a sealed 250ml bottle in shaking water. During this 
step, non-resistant starch will be solubilized and hydrolyzed to glucose and maltose by the 
combined action of the two enzymes. The reaction will then be terminated by PH adjustment 
and temporary heating. Protein in the sample will be digested with protease. For the 
measurement of high molecular weight dietary fiber, Ethanol or industrial methylated sprits 

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will be added and the insoluble and perceptible soluble dietary fiber will be captured, washed 
with ethanol and acetone, dried, and weighed. The final result will then be obtained by using 
the formula: Total dietary fiber (%) = (HMWDF+LMWSDF)/1000 (HMWDF = high 
molecular weight soluble dietary fiber LMWSDF= low molecular weight soluble dietary 
fiber) 
6.2.Urine iodine analysis 
  
Analytical methods consist of an initial digestion step to get rid of interfering substances, 
followed 
by 
the 
Sandell–Kolthoff 
reaction, 
of 
which 
the 
reading 
is 
obtained 
spectrophotometrically or colorimetrically. The digestion step will be held to remove Interfering 
substances in urine. The sample will be digested using the appropriate strong acid (e.g. 
ammonium persulphate). The oxidizing step will be carried out in a heated environment. The 
Sandell–Kolthoff reaction is a two-step reaction in which the rate of reaction will be measured 
during the reduction of the yellow-coloured ceric ions by arsenic in the presence of iodide to 
form colourless cerous ions and elemental iodine.  
6.3.Statistical analysis plan 
Given the variability of the type of data generated from each assessment, data analysis and 
interpretation shall be done in the respective the nature of each data. 
Anthropometric measurement data shall be analyzed using corresponding index of Weight for 
height, height for age, weight for age and MUAC for age and interpreted using international 
standards. ENA 2020 shall be used to record the measurements and convert to respective Z-
scores.     
The Data generated from CARS diagnosis will be on a scale from one (normal behavior) to four 
(severely abnormal behavior) for each category of questions. The scores between 30 and 37 
indicate mild to moderate ASD, while scores between 38 and 60 indicate severe ASD. These 
results shall be analyzed by SPSS version 26 for descriptive statistics and correlations  
In case of urine iodine analysis, the decrease in yellow color over a fixed period is measured by a 
colorimeter with an appropriate filter or with a spectrophotometer and plotted against a standard 

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curve constructed with known amounts of iodine. The resulting reading of iodine content shall 
then be compared among case and control groups of the study. SPSS version 26 shall be used to 
for descriptive statistics, significant difference and correlations   
The data generated from proximate analysis of food samples shall be recorded for each analysis 
and calculated for average. The final figure represents the amount of nutrient present per 100 gm. 
thus amounts consumed by children. This data shall then be compared among case and control 
groups of the study. SPSS version 26 will also be used for descriptive statistics, significant 
difference and correlations