the canadian journal of clinical nutrition (cjcn) is the...

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The Canadian Journal of Clinical Nutrition (CJCN) is the official

journal of Association of Editors, and is published by Global Science

Heritage, Toronto, Canada. Global Science heritage is a registered

publisher by the Library and Archives/ Government of Canada

(www.collectionscanada.gc.ca), Business License Number: 220080600.

Statements printed in this journal are those of the authors and no

liability can be accepted by the Global Science Heritage and Editorial

Board for any information in the published materials, although believed

to be accurate and reliable. Any reproduction or use of contents of the

journal is permitted only if the source is properly cited.

iii

Editorial Board

Editors

Allal Ouhtit, MSc, PhD, Department of Genetics, College of Medicine and Health

Sciences, Sultan Qaboos University, Oman

Mostafa Waly, MSc, MPH, PhD, Department of Food Science and Nutrition,

College of Agricultural and Marine Sciences, Sultan Qaboos University, Oman

Yahya Al-Farsi, MD, MPH, Dr.PH, Department of Family Medicine and Public

Health, College of Medicine and Health Sciences, Sultan Qaboos University,

Oman

Managing Editors

Neeru Bhatt, MSc, PhD, Associate

Research Fellow, Department of Food

Science and Nutrition, College of

Agricultural and Marine Sciences, Sultan

Qaboos University, Oman

Azza Abd El-Moneim Attia Mohamed,

MSc, PhD, Lecturer of Histology, Ain

Shams University, Cairo, Egypt

Eman Ahmed Mahmoud Mahmoud El-

Shabasy, MSc, PhD, Lecturer of

Biology, Zoology Department, Faculty of

Science, Mansoura University, Egypt

Hamza M. Abu-Tarboush, MSc, PhD, Professor, Food Science and Nutrition

Department, College of Agriculture, King

Saud University, Riyadh, Saudi Arabia

Hassan I.H. El-Sayyad, MSc, PhD, Professor of Experimental Embryology,

Zoology Department, Faculty of Science,

Mansoura University, Egypt

Rajiv Lochan Gaur, PhD, Department

of Pathology, Stanford Blood Center,

Stanford University, Palo Alto, USA

Syed Tasleem Raza, MSc, PhD, Assistant Professor and Head Molecular

Diagnosis in Biochemistry Department,

ERA‘S Lucknow Medical College,

Lucknow, India

Zafar Iqbal, MPhil, PhD, Assistant

Professor of Genetics, King Saud Bin

Abdulaziz University of Health Sciences,

National Guards Health Affairs, Riyadh,

Saudi Arabia

Administration

Ishita Gupta, MSc, Department of Genetics, College of Medicine and Health Sciences,

Sultan Qaboos University, Oman

iv

Advisory Board

Ali, Amanat, MSc, PhD, Associate

Professor, Department of Food Science

and Nutrition, College of Agricultural

and Marine Sciences, Sultan Qaboos

University, Oman

Ali, Badreldin H, BVSc, PhD, Professor,

Department of Pharmacology and

Clinical Pharmacy, College of Medicine

and Health Sciences, Sultan Qaboos

University, Oman

Al-Farsi, Khalil, MD, PhD, Consultant,

Department of Hematology, College of

Medicine and Health Sciences, Sultan


Al-Khaduri, Maha, MD, PhD,

Consultant, Department of Obstetrics and

Gynecology, College of Medicine and

Health Sciences, Sultan Qaboos

University, Oman

Al-Sabah, Haleama H, MSc, PhD,

Assistant Professor, Department of

Public Health, Faculty of Medicine, Al-

Najah National University, Palestine

Al-Shafaee, Mohamed, MD, Dr.PH,

Associate Professor, Department of

Family Medicine and Public Health,

College of Medicine and Health

Sciences, Sultan Qaboos University,

Oman

Al-Sharabati, Marwan, MD, Dr.PH,

Associate Professor, Department of

Behavioral Medicine, College of



Al-Sinani, Siham, MD, Consultant,

Department of Gastroenterology, Sultan

Qaboos University Hospital, Oman

Al-Zadjali, Majed, MD, MSc, Director,

Department of Malaria Eradication,

Ministry of Health, Oman

Arafa, Mostafa, MD, MPH, Dr.PH,

Professor, Cancer Research Unit, King

Saud University, Riyadh, Saudi Arabia

Ausman, Lynne, PhD, Professor, Jean

Mayer USDA HNRCA, Tufts University,

Boston, USA

El-Badawy, Amal, MSc, PhD, Assistant

Professor, Department of Fundamentals

and Administration Nursing, College of

Nursing, Sultan Qaboos University,

Oman

Farhat, Mohamed F, MPH, DrPH,

Professor, Department of Nutrition, High

Institute of Public Health, Alexandria

University, Egypt

Gad, Ashry, MD, DrPH, Professor,

Department of Epidemiology, High

Institute of Public Health, Alexandria

University, Egypt

Hussein, Izzeldi, PhD, Micronutrients

Specialist, Institute of Brain Chemistry

and Human Nutrition, London, United

Kingdom

Kilani, Hashim, PhD, Professor,

Department of Physical Education,

College of Education, Sultan Qaboos

University, Oman

Mo‘ez Al-Islam E.M. Faris, MSc, PhD,

Assistant Professor, Department of

Nutrition, Faculty of Pharmacy and

Medical Sciences, Petra University,

Amman, Jordan

v

Musaiger, Abdulrahman O, MPH, DrPH,

Professor of Human Nutrition, Head of

Arab Center for Nutrition Research,

Bahrain

Obeid, Omer, MD, PhD, Professor,

Department of Nutrition and Food

Science, American University, Beirut,

Lebanon

Sabra, Amr A, MD, MPH, Dr.PH,

Professor, Department of Primary Health

Care, High Institute of Public Health,

Alexandria University, Egypt / Assistant

Professor & Consultant Family and

Community Medicine Department,

College of Medicine, University of

Dammam, Saudi Arabia

Saleh, Jumana, PhD, Associate Professor,

Department of Clinical Biochemistry,

College of Medicine and Health

Sciences, Sultan Qaboos University,

Oman

Wessig,Volkmar, PhD, Associate

Professor, Department of Pharmaceutical

Sciences College of Pharmacy Glendale,

Midwestern University, USA

Yousef, Randa, MD, DrPH, Associate

Professor, Department of Family

Medicine and Public Health, College of



vi

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ix

Table of Contents

Editorial

Ouhtit A, Gupta I, Sheikh Z, Shanmuganathan S, Al-Farsi Y, Waly MI, Raj HG M. Nutrigenomics

and Complementary Alternative Medicine. Canad J Clin Nutr 2013; 1 (1): 1-4.

Research Articles

Janbi OS, Sabra AA, Sebiany AM, Hafez AS. Predictors of Maternal Weight Gain during Normal

and High Risk Pregnancies: A Retrospective Study in Primary Care, Eastern Saudi Arabia. Canad J

Clin Nutr 2013; 1 (1): 5-15.

Rady M, Al-Muslemani M, Salama R. Determinants of Overweight and Obesity among Qatari

Children (2-5 years) in Doha, Qatar-2010. Canad J Clin Nutr 2013; 1(1): 16-26.

Gad A, AL-Quaiz J, Khoja T, As-Sharif A, Al-Manea H, Al-Edriss A, Shafi S. Anemia among

Primary School Children (5 -12 years) in Riyadh Region, Saudi Arabia: A Community-Based Study.

Canad J Clin Nutr 2013; 1(1): 27-34.

Mohamed BA, Alhamdan AA, Samarkandy MM. Dietary Practice and Physical Activity in Children

with Down Syndrome and Their Siblings in Saudi Arabia. Canad J Clin Nutr 2013; 1(1): 35-46.

Mohamed AA, Saad MM, Abdeen SH, Marei MK. Generation of Insulin Producing Cells using

Mesenchymal Stem Cells Derived from Bone Marrow of New-Zealand White Rabbits. Canad J Clin

Nutr 2013; 1(1): 47-66.

Waly MI, Al-Farsi YM, Ouhtit A. The Role of Folate on Colorectal Cancer in the Arabian Gulf

Cooperation Council Countries: An Overview. Canad J Clin Nutr 2013; 1(1): 67-73.

Letter to the Reader

Waly MI. Should Children with Autism Continue Taking Folic Acid Supplement? Canad J Clin Nutr

2013; 1(1): 74.

The Canadian Journal of Clinical Nutrition, Volume 1/Number 1, January 2013 ISSN 1927-8942 (Print Edition), ISSN 1927-8950 (Online Edition)

Canad J Clin Nutr is published by Global Science Heritage (http://www.globalscienceheritage.org), a registered publisher by the Library and Archives/Government of Canada, Business License Number: 220080600 (www.collectionscanada.gc.ca).

Pag

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Nutrigenomics and Complementary Alternative Medicine

Allal Ouhtit1, Ishita Gupta

1, Zoya Sheikh

1, Somya Shanmuganathan

1, Yahya Al-Farsi

2,

Mostafa I. Waly3,4

, Madhwa HG Raj5*

1Department of Genetics,

2Department of Family Medicine and Public Health,

3Department

of Food Science and Nutrition, Sultan Qaboos University, Muscat, Oman 4Department of Nutrition, High Institute of Public Health, Alexandria University, Egypt

5Department of Obstetrics and Gynecology, Louisiana State University Health Sciences

Center, New Orleans, Louisiana, USA

*Corresponding Author: Dr. Madhwa HG Raj. Email: [email protected]

ABSTRACT

Nutrigenomics applied high-throughput ‗omics‘ techniques in nutrition research to enable

investigation into interactions between nutrients with the genome at a molecular level. One of

the emerging areas of research in nutri-genomics includes Complementary Alternative

Medicine (CAM), which can be used to treat various diseases including type2 diabetes,

cancer and obesity. Research in CAM includes identification of the active compounds present

in various herbal and dietary products, and evaluating these compounds for their effects on

human health. Only few studies have explored the effects of these compounds when used in

synergistic, additive or antagonistic combinations. One of the striking features of CAM is the

low toxicity of natural compounds used as supplements. However most of the active

ingredients are not ―hydrophyllic‖ but are ―lypophyllic‖, resulting in limited absorption from

GI tract, when ingested orally. This has led to limited bioavailability and it needs application

of innovative techniques to overcome this problem. We have evaluated combination therapy

and its effects as a solution to this problem. This report will discuss general concepts in

nutrigenomics related to CAM, effects of combination therapies and possible mechanisms of

action that come in to play with combination therapies, with particular focus on

phytocompounds used as anti-cancer agents.

Key words: Nutrigenomics, Phytochemicals, CAM, Cancer.

Ouhtit A, Gupta I, Sheikh Z, Shanmuganathan S, Al-Farsi Y, Waly MI, Raj HG M.

Nutrigenomics and Complementary Alternative Medicine. Canad J Clin Nutr 2013; 1 (1): 1-4.

Nutrigenomics studies the effects of food

and its constituents at molecular level so as

to gain insight into the mechanisms of

interaction between nutrients and other

dietary bioactives with the genome.

Nutrigenomics aims to understand the

body‘s response to different types of diets

and food through various ‗omics‘

techniques including transcriptomics,

proteomics and metabolomics, although

the most common technique used is

transcriptomic analysis (1). Tools used to

measure the transcriptome are well

developed including gene expression

microarray profiling, single nucleotide

polymorphisms (SNPs) and genotyping. In

contrast, tools to measure the proteome

and metabolome are less developed

including techniques such as gel

electrophoresis, mass spectophotometry,

nuclear magnetic resonance imaging, and

chromatography (2); This technique has

provided a considerable amount of data on

several factors such as novel function of

food factors, the unknown mechanism of

the effect of nutrients, and even safety

Page 1-4




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issues of foods (3). Nutrigenomics is

emerging as a new competent field in

research that integrates genetics and

dietary recommendations to study protein

expression and metabolite production.

Nutrigenomics can help improve our

understanding of how nutrition influences

metabolic pathways and homeostatic

control, which can be used to discover

naturally occurring chemical agents in

food to help prevent the onset of diseases

such as obesity, type-2 diabetes and

cancer. Nutrigernomics, also involves

determining certain genes and markers

during the early phase of diet-related

diseases. Once such a marker or a gene is

identified and measured in an individual,

the degree to which they can be

susceptible to the onset of that particular

disease can be quantified and a

personalized dietary recommendation can

be established for the individual.

Nutrigenomics aims to elucidate the effect

of bioactive food compounds on health

that can lead to the development of

functional foods that keep people healthy

according to their individual needs. One

area of particular research interest in

nutrigenomics can include Complementary

Alternative Medicine (CAM). Recently,

research in CAM includes identification of

the active compounds present in various

herbal and dietary products, and evaluating

their anti-cancer properties. Polyphenols

from green tea, grape seed/skin,

anthocyanin and pigments from many

flowers, algae, fruits and vegetables are

some of the compounds that have been

tested. A common property of many of

these compounds is their anti-oxidant/free

radical scavenging ability. However, some

compounds preferentially induce high free

radical formation selectively in cance

cells, to cause growth inhibition and death

of cancer cells without affecting normal

cells.

Extensive studies on CAM have focused

on effects of individual compounds

derived from herbs/plants, using

concentrations that are typically higher

than their ‗bioavailable‘ concentration (the

serum levels achieved by oral intake of

extracts, as practiced in CAM). However,

only few studies explored the effects of

these compounds when used in synergistic,

additive or antagonistic combinations; and

each of the individual chemicals used in a

combination could target multiple

signaling pathways in the cancer cell. One

of the striking features of oral

administration of CAM was the low

toxicity and showed effects on various

types of cancers. The synergistic and / or

additive mechanisms that come into play

during combination therapies result in

reducing the ―effective dose‖ to

‗bioavailable‘ levels. Thus while

individual compounds are not effective by

themselves, combination becomes

effective.

In collaboration with Prof. Madhwa Raj‘s

group (the Louisiana State University,

New Orleans, Louisiana), and based on

the idea of Synergism theory we

performed extensive studies where we

tested the effects of the combination of 2

compounds, Indole-3-carbinol (I3C) and

Resveratrol, each used at ‗bioavailable’

levels, on SK-OV-3 ovarian cancer cells

(3). SK-OV-3 cells were treated with

various doses of I3C, RE or I3C+RE and

proliferation assay was used to examine

cell growth. The study revealed that

I3C+RE synergized to induce a higher

number of cell death than each of these

compounds used individually (3).

Analysis of apoptosis-associated genes

revealed inhibition of Retinoblastoma

protein (Rb) and Survivin (SVV) gene

expression levels; this was accompanied

by elevation of p21, a p53-downstream

transcriptional target gene (3). Cell cycle

was inhibited at both G1 and G2/M by

individual treatments, and accentuated by

a combination. ELISA revealed that

while, CA125 was inhibited by either I3C

or RE treatments, basal nitric oxide

production was inhibited by I3C and I3C



Pag

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+ RE but not RE alone (3), in addition

microarray analysis was performed and

we have identified several unique genes

that are overexpressed as a result of

combination therapy (manuscript in

preparation).

Ongoing studies in our laboratories are

testing the combination of dozens of

other compounds in several cancers In

addition; selected phytochemical

combinations will be tested to validate

their anti-cancer effects in vivo using

xenograft model of breast cancer (4).

Future work will include the

establishment of a powerful combination

of compounds (each individual

compound is used at its bioavailable

concentration in a synergistic/additive

manner) that can potentially induce a

maximal cell growth inhibition/cell death

in vitro and in vivo. Furthermore, in

addition to understanding the underlying

molecular mechanisms of the

combination, these studies will lead to the

identification of potential biomarkers or

candidate gene targets to guide the design

of anti-cancer therapeutic strategies,

follow progress of therapies as well as in

early diagnosis.

REFERENCES

1. Kato H. Nutrigenomics: The

cutting edge and Asian

perspectives. Asia Pac J Clin Nutr

2008; 17 (1):12-15.

2. Hesketh J. Personalised nutrition:

How far as nutrigenomics

progressed?. Europ J Clin Nutr

2012 [Epub ahead of print].

3. Raj M, Abd-Elmageed ZY, Zhou J,

Gaur RL, Nguyen L, Azam GA,

Braley P, Rao PN, Fathi IM, Ouhtit

A. Synergestic action of dietary

phyto-antioxidants on survival and

proliferation of ovarian cancer

cells. Gynecological Oncology

2008; 110(3): 432-438.

4. Ouhtit A, Abd Elmageed YZ,

Abdraboh M, Lioe

TF, Raj MG. In

vivo evidence of a direct role of the

standard form of CD44 in breast

metastasis. Am J Pathol (In press)



Pag

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Table 1: Bioactive compounds and their mechanisms of action

Source Active

Ingredient

Literature

Broccoli

-Indole-3-

Carbinol

(I3C)

I3C induces apoptosis, inhibits cell growth and has antiangiogenic

activities

I3C induces G1 cell cycle arrest and inactivates Akt

I3C inhibits activation of transcription factors including nuclear factor-

kappa B, SP1, estrogen receptor, androgen receptor and nuclear factor-E2-

related factor 2 (Nrf2)

Grape skin

and seeds

Resveratrol

(RE)

RE interferes with AKT, enhances p53, and induces apoptosis

RE exhibits Cox-1 inhibitory activity, and causes G1 arrest

RE induces apoptosis by TRAIL sensitization and down regulates

survivin expression

RE possesses vasorelaxing, anti-inflammatory, anti-lipidemic, anti-

estrogenic, antioxidant, anti-fungal and antibacterial properities

Tea

Epigallo-

Catechin

Gallate (ECG)

ECG inhibits Hsp90 function, hypoxia and serum induced HIF-1 alpha

protein accumulation, and VEGF expression

ECG enhanced responses induced by curcumin on breast cancer cells

Spirulina

Phycocyanin

(PC)

P inhibits cell proliferation and apoptosis in different cancer cell lines

P inhibits MDR1 through reactive oxygen species and cyclooxygenase-2

mediated pathways

Turmeric

roots

Curcumin

(CUR)

CUR binds to a number of proteins and inhibits the activity of various

kinases, induces apoptosis, and has anti-proliferative effect

CUR regulates expression of inflammatory enzymes, cytokines, adhesion

molecules and cell survival proteins

CUR down regulates cyclin D1, cyclin E, MDM2 and up regulates tumor

suppressors p21, p27 and p53

CUR exhibits antioxidant, anti-inflammatory, antiviral, antibacterial,

antifungal and anticancer activities

Stamens of

Saffron

Crocin (Cr) Saffron causes apoptosis inhibits DMBA-induced skin carcinogenesis

Saffron and its main constituents, such as crocusatin H, crocin-1 and

crocin-3 have anticancer and anti-tumour activities

Plant food

Quercetin

(Querc)

Quercetin downregulates mutant p53 in BC cells leading to G1 phase

arrest of cell cycle, inhibits tyrosine kinase, both in- vitro and in- vivo

A number of these actions have also been demonstrated in ovarian cancer

cells via inhibition of heat shock protein 70

It sensitized cisplatin in inhibiting proliferation of ovarian cancer cells

It increased TGF-β and inhibited OVCAR-433 ovarian cancer cell

proliferation, as administration of a monoclonal antibody to TGF-β

reversed these effects



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Predictors of Maternal Weight Gain during Normal and High Risk

Pregnancies: a Retrospective Study in Primary Care, Eastern Saudi Arabia

Olfat S. Janbi 1, Amr A. Sabra

1,2*, AbdulAziz M. Sebiany

1, Ahmed S.Hafez

1,3

1Department of Family and Community Medicine, College of Medicine, University of

Dammam, Saudi Arabia. 2

Primary Health Care Divisions, High Institute of Public Health,

Alexandria University, Egypt. 3Department of Community, Environmental and Occupational

Medicine, Faculty of Medicine, Ain-Shams University, Egypt

*Corresponding Author: Professor Amr Ahmed Sabra. Email: [email protected]

ABSTRACT

Background: Pregnancy is the only time in a woman's life where weight gain is expected and

encouraged. Therefore, women are concerned whether pregnancy will have an impact on their

body weight. Objective: To determine gestational weight gain during normal and high risk

pregnancy and to find-out the maternal factors that are associated with maternal weight gain

during normal and high risk pregnancies. Subjects and Methods: A retrospective study was

conducted in Al-Khobar city, Eastern Saudi Arabia. All pregnant women registered, during the

year 2010-2011 (n=323), at three randomly selected primary health care centers were included in

the study. The number of records with completed registered studied variables and weight gain

during pregnancy was 229 records (70.9% of total records) was divided according to the criteria

of high risk pregnancy, into two groups, namely normal pregnancy (n=123) and high risk

pregnancy (n=106). The maternal body weight gain was divided into three categories as low

weight gain (≤ 8.0 kg), normal weight gain (8.1—16.0 kg) and high weight gain (≥ 16.1 kg).

Statistical analysis was done using descriptive and analytic statistics as well as logistic regression

analysis. Results: The rate of women having normal maternal weight gain during normal

pregnancy was 35% as compared to those 18.9% during high risk pregnancy. While, 63.4% of

women had low weight gain during normal pregnancy, 80.2% of them had low weight during

high risk pregnancy. About 31.2% of women during normal pregnancy were aged 35-45years old

when compared to 68.8% of women during high risk pregnancy and the difference was

statistically significant (p<0.01). Increased parity and number of living children are significantly

associated with low maternal weight gain during normal pregnancy. Decreased antenatal visits

during high risk pregnancy were significantly associated with low weight gain during high risk

pregnancy. Conclusion: Low weight gain was more during high risk pregnancy; increase in

parity and number of living children during normal pregnancy, decreased antenatal visits during

high risk pregnancy are significantly associated with low maternal weight gain. Suitable measures

should be taken to control maternal body weight gain during pregnancy.

Key words: Maternal Weight Gain, Primary Health Care, Predictors, Saudi Arabia.

Janbi OS, Sabra AA, Sebiany AM, Hafez AS. Predictors of maternal weight gain during

normal and high risk pregnancies: a retrospective study in primary care, Eastern Saudi Arabia.

Canad J Clin Nutr 2013; 1 (1): 5-15.

INTRODUCTION

Pregnancy is the only time in a woman's

life where weight gain is expected and

encouraged. Therefore, women are

concerned whether pregnancy will have an

impact on their body weight. There are

many body changes during pregnancy: an

inevitable one is that pregnancy and

weight gain go hand in hand, not making it

any easier for the body image during

pregnancy. Maternal weight gain in

pregnancy can serve as a good mean of

assessing the wellbeing of the pregnant

mother (1). The weight of the growing

fetus itself should expect to have a gain

between 10-12 kg and a 12-15kg of ‗baby‘

respectively with a single pregnancy.

During the first trimester pregnancy

Page 5-15



Pag

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weight gain should be between 1-2 kg, and

then about 500g per week for the rest of

the pregnancy term (2).

Inadequate prenatal weight gain and low

weight gain during pregnancy are

significant risk factors for intra-uterine

growth retardation, pre-term delivery and

low birth weight in infants (3-7).

Excessive weight gain on the other hand

can lead to adverse maternal and fetal

outcomes (8,9). Accordingly, suggestions

for optimal weight gain is needed to ensure

the best outcomes (10- 12). Gestational

weight gain was defined as the difference

between the maternal weight measured

within one week prior to delivery and the

maternal weight recorded at the first visit

to the hospital. Gestational weight gains

were grouped into three categories as low

weight gain (≤ 8.0 kg), normal weight gain

(8.1—16.0 kg) and over weight gain (≥

16.1 kg) (13).

Excessive gestational weight gain, in

Saudi Arabia, is emerging as an important

predictor of maternal and offspring

obesity, as well as obstetrical

complications. Independent of their weight

entering pregnancy, mothers who gain

excessively during pregnancy are more

likely to deliver by cesarean section, have

an unsuccessful trial of labor after

cesarean section, develop pre-eclampsia,

retain excessive weight after delivery, and

become overweight or obese in later life

(14). Various studies reported that the

socio-demographic predictors of excessive

weight gain during pregnancy were null

parity, pre-pregnancy overweight, body

mass index, low socioeconomic status, and

young maternal age (2,4,6,8,14). The

nature of weight gain during normal and

high risk pregnancy in Saudi Arabia

remains undetermined. The aim of the

present study was to determine gestational

weight gain during normal and high risk

pregnancy and find-out the maternal

factors associated with weight gain during

normal and high risk pregnancies.

SUBJECTS AND METHODS

A retrospective epidemiological

investigation based on population

background was conducted during the year

2012 in Al-Khobar city, part of the eastern

province in the Kingdom of Saudi Arabia

(KSA). Married fertile women having

children in catchments of randomly

selected primary health care centers

(PHCCs) were the units of observation and

the target population of the present study.

All available medical records of pregnant

women in three out of the eight PHCCs, in

Al-Khobar city, were randomly selected

namely; Al-Akrabia, Ibn Hayyan and Al-

Bayonia PHCCs. The specific inclusion

criteria of the present study were the

presence obstetric records of Saudi and

Non Saudi women that attended their first

antenatal visit during the study period in

the selected PHCCs. The records of all

pregnant women, who were developed

according to the World Health

Organization (WHO) known as "Mother

and child health passport", who started

their antenatal care from 1st October 2010

to 30th September 2011 were collected

from the three randomly selected PHCCs.

The total number of these antenatal

records was 323, and the number of

records with complete registered studied

variables and gained weight during

pregnancy was 229 records which

represented 70.9% of the total records;

they were divided according to the criteria

of high risk pregnancy,(15) into two

groups, namely normal pregnancy (n=123)

and high risk pregnancy (n=106). The

available maternal variables in the present

study were socio-demographic and

reproductive characteristics as well as

family, past-medical and surgical histories,

current obstetric history and maternal body

weight gain. Obstetric outcome included

preeclampsia, eclampsia, gestational

diabetes, ante partum hemorrhage, full

term and postdate deliveries. Values of

maternal body weight gain were divided

into three categories as low weight gain (≤



Pag

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8.0 kg), normal weight gain (8.1—16.0 kg)

and high weight gain (≥ 16.1 kg) (13).

Demographic, antenatal data were

explored; and women with normal weight

were used as the reference or the

comparison group for data analysis. The

socio-economic status of the mothers was

determined by scoring system (16) using

parental education (0-4 scores for either

paternal or maternal education); Paternal

occupation (2-7 scores) and maternal

occupation (0-1 score). Accordingly, the

total score of socio-economic status ranged

from 2-16 scores.

Data entry and verification was done and

variables were described using frequency

distribution for categorical variables. The

chi-square (χ2) and Fishers Exact test were

used as a test of significant values for

comparison of categorical variables and t

test was used as a test of significance for

quantitative variables. P value < 0.05 was

chosen as the level of statistical

significance using Statistical Package of

Social Sciences (SPSS) version16 (17).

Logistic regression analysis was done to

determine the main factors associated with

weight gain and the results were expressed

as Odds Ratio (OR) and corresponding

95% as confidence interval (CI). Pilot

study was applied on fifteen files in Al-

Akrabia PHCC in order to test the validity

and reliability. Permission was taken from

the concerned authority to conduct the

study and confidentiality of the

information was strictly adhered, with data

to be used only for research purpose.

RESULTS

The total registered records of women who

attended antennal care during the study

period were 323 records and 29.1% were

excluded due to lack of registration of

some variables under the study. A total of

229 maternal antenatal records were

enrolled in the study. They were classified

into normal (n=123) and high risk

pregnancy (n=106) groups according to

the criteria of high risk pregnancy15. The

criteria of high risk pregnancy were

previous stillbirth or neonatal deaths (4%),

isoimmunisation RH (5.9%), previous

surgery on reproductive tract (14.2%),

history of low birth weight (< 2500g) in

last pregnancy (7.7%), and any medical

disease or condition (23.2%) (Table 1).

Maternal weight gain was normal in 35%

women during normal pregnancy as

compared to 18.9% of the women during

high risk pregnancy. However, low weight

gain during normal pregnancy was

observed among 63.4% women during

normal pregnancy when compared to

80.2% during high risk pregnancy. The

overweight gain was approximately the

same during normal and high risk

pregnancies (Figure 1). During normal

pregnancy 31.2% of women were in the

age group of 35-45years old compared to

68.8% during high risk pregnancy and the

difference was statistically significant

(p<0.01) (Table 2). Moreover, there was

no statistical significant difference

between the studied women during normal

and high risk pregnancies regarding

nationality and maternal occupation. The

reproductive characteristics of studied

women revealed that 47.5% of them

during normal pregnancy had abortions

compared to 52.5% who had undergone

abortion during high risk pregnancy; and

28.1% of women during normal had 5 or

more living children as compared to 71.9%

of women during high risk pregnancies.

(Table 2). Family history of women

showed that diabetes mellitus,

hypertension, multiple pregnancy, and

consanguinity are nearly the same during

normal and high risk pregnancy (Table 3).

Increase parity and increased number of

living children are significantly associated

with low maternal weight gain during

normal pregnancy (Table 3). However,

decreased antenatal visits during high risk

pregnancy were significantly associated

with low maternal weight gain during high

risk pregnancy (Table 4). Moreover, an

association between maternal weight gain,



Pag

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obstetric outcomes and prenatal

morbidities was determined; however, the

number of women during normal

pregnancy was too small. Logistic

regression analysis revealed that mother‘s

education (OR= 1.7, CI =1.12- 2.45,

Model X2 (15) = 19.95, P<0.5) was the

only significant (P<0.05) predicting factor

associated with maternal weight gain

during normal pregnancy (Table 4).

However, during high risk pregnancy

neither socio-demographic factors nor

reproductive characteristics were

predictors of maternal weight gain.

DISCUSSION

Maternal weight was recorded at almost

all antenatal primary care attendees and

weight gain was considered as a clinical

test during antenatal care. The amount of

maternal weight gain was found to be

compatible with normal pregnancy

outcome (18-20).These have led to studies

resulting in recommended weight gain

during pregnancy (21). In the present study

most of the women studied in Saudi were

aged 25-35 years (56%); 94.1% were

Saudi nationals and 95% were housewives.

The findings are similar to the study done

among Qatari married women (15), but a

higher value for maternal age was reported

by a study in Saudi Arabia (23). The

reproductive characteristics of Saudi

women showed that 26.6% had abortion

and 34.1% had up to two children; this

finding is inconsistent with other studies.

(6,8,13,22). Past-medical history of

anemia was observed among 20.4%,

14.2% had past surgical history of

cesarean section, 27.5% had normal body

weight gain (8.1 to <16) during pregnancy

and the mean antenatal visits was 2.5±1.2.

Data of other previous studies (3, 13, 15,

23, 24) were inconsistent with these

findings in the present study. The criteria

of high risk pregnancy were previous

stillbirth or neonatal deaths (4%),

isoimmunisation RH (5.9%), previous

surgery on reproductive tract (14.2%),

history of low birth weight in last

pregnancy (7.7%), and any medical

disease or condition (23.2%). This study

showed that there was statistically

significant (P<0.01) difference in age

distribution of studied women during

normal and high risk pregnancies.

However, there was no statistical

significant difference between the studied

women during normal and high risk

pregnancies regarding nationality and

maternal occupation.

Also the reproductive characteristics of

studied women revealed that there was

statistical significant (P<0.01) difference

between the studied women during normal

and high risk pregnancies number of living

children. Family history of women showed

that diabetes mellitus, hypertension,

multiple pregnancy, and consanguinity are

similar during normal and high risk

pregnancy. Moreover, 18.8% of women

studied during normal pregnancy had past-

medical history of anemia, 12.5% had

past-surgical history of caesarean section,

56.9% had 3-4 antenatal visits, 60%

delivered pre-term babies and 68.8% had

normal maternal weight gain during

normal pregnancy.

Excessive gestational weight gain in Saudi

Arabia is emerging as an important

predictor of maternal obstetric

complications. Mothers who gain weight

excessively during pregnancy are more

likely to deliver by caesarean section,

develop pre-eclampsia, and become

overweight or obese in later life (14).

Increase parity and increased number of

live births are significantly associated with

low maternal weight gain during normal

pregnancy. However, decreased antenatal

visits during high risk pregnancy were

significantly associated with low maternal

weight gain during high risk pregnancy.

These finding are inconsistent with

findings of other studies performed in

Ghana (2), Africa (25), Saudi Arabia

(23,24) Also, there is an association

Page 7-17



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between maternal weight gain and either

obstetric outcomes or prenatal morbidities,

although the present study involved small

number of women in each variable under

investigation during normal pregnancy.

Logistic regression analysis revealed that

mother‘s education (OR= 1.7, CI =1.12- 2.

45) (P<0.05) was the only significant

predicting factor associated with maternal

weight gain during normal pregnancy.

However, none of the socio-demographic

factors and reproductive characteristics

was found to be predictors of maternal

weight gain during high risk pregnancy.

This is not in-accordance with various

studies, which reported that an antenatal

visit during pregnancy was the significant

preventive factor against adverse

pregnancy outcome and complications

(11-15).

CONCLUSION

The results of the present study

demonstrated that 29.1% of the records

were incomplete and was missing

important data which may have not been

performed or not recorded. Low maternal

body weight gain during normal pregnancy

was significantly associated with increase

parity and increased number of children.

However, decreased antenatal visits during

high risk pregnancy were associated only

significantly with low maternal weight

gain. Also there is an association between

maternal weight gain and obstetric

outcomes and prenatal morbidities.

Logistic regression analysis demonstrated

that mother‘s education was the only

significant predicting factor associated

with maternal body weight gain during

normal pregnancy. From the results of the

present study, it is recommended that all

primary health care team members

providing antenatal care services should

receive continuous medical education and

in-service training about the new WHO

model and be aware about the principles

and importance of proper registration and

recording. Health education sessions

should be conducted for all females with

particular attention to pregnant women

about the hazards associated with

excessive body weight gain during

pregnancy and the different methods of its

control, with special emphasis on lifestyle

modification.

Conflicts of Interest

The authors indicated no potential or

actual conflict of interest pertaining to this

study.

Authors' Contributions

All authors made full contribution to data

acquisition, interpretation of results,

drafting and revising the final manuscript.

All authors read and approved the final

manuscript.

Study Limitations

There were no limitations.

Acknowledgements

The authors deeply extended their

appreciation to the study subjects for their

dedicated effort to complete this study.

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1. Varma TR. Maternal weight and

weight gain in pregnancy and

obstetric outcome. Int J Gynaecol

Obstet 1984; 22(2):161-166.

2. Addo VN. Body mass index,

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3. Lawoyin TO. Maternal weight and

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4. Tulman L, Morin KH, Fawcett J.

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functional status, symptoms, and

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5. Suitor CW. Maternal Weight Gain:

A Report of an Expert Work

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Center for Education in Maternal

and Child Health.1997

6. Marsoosi V, Jamal A, Eslamian L.

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pregnancy weight gain, and

preterm delivery. Int J Gynaecol

Obstet 2004; 87(1):36-37.

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Scanlon KS, Perry G, Ferre C,

Blackmore-Prince C. Pre-

pregnancy body mass index and

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8. Kumari AS. Pregnancy outcome in

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107.

9. Ekblad U, Grenman S. Maternal

weight, weight gain during

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39(4):277-283.

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during pregnancy: Colorado

Pregnancy Risk Assessment

Monitoring System (PRAMS),

1997-2000. Colorado. Health

Statistics Section, Colorado

Department of Public Health and

Environment, 2003.

11. Abrams B, Altman SL, Pickett KE.

Pregnancy weight gain: still

controversial. Am J Clin Nutr

2000; 71:1233S-1241S.

12. Bracero LA, Byrne DW. Optimal

maternal weight gain during

singleton pregnancy. Gynecol

Obstet Invest 1998; 46(1):9-16.

13. Cedergren M. Effects of gestational

weight gain and body mass index

on obstetric outcome in Sweden.

Int J Gynaecol Obstet 2006;

93(3):269-274.

14. Stuebe AM, Oken E, Gillman MW.

Associations of diet and physical

activity during pregnancy with risk

for excessive gestational weight

gain. Am J Obstet Gynecol 2009;

201(1):58-61.

15. World health organization. WHO

Antenatal Care Randomized Trial:

Manual for the Implementation

of the New Model.

WHO/RHR/01.30. WHO, Geneva,

2002.

16. Park K. Demography and family

planning: Textbook of preventive

and social medicine.20th ed.

Jabalphurs: M/s Banarasidas

Bhanos publisher 2009 pp.411-

446.

17. Statistical Package for Social

Sciences (SPSS) version 16 for

Windows. 2007. SPSS Inc., 1989-

2007.

18. Copper RL, DuBard MB,

Goldenberg RL, Oweis AI. The

relationship of maternal attitude

toward weight gain to weight gain

during pregnancy and low birth

weight. Obstet Gynecol 1995;

85(4):590-595.

19. Crane JM, White J, Murphy P,

Burrage L, Hutchens D. The effect

of gestational weight gain by body

mass index on maternal and

neonatal outcomes. J Obstet

Gynaecol Can 2009; 31(1):28-35.

20. Rosso P. A new chart to monitor

weight gain during pregnancy. Am

J Clin Nutr 1985; 41(3):644-652.

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21. National Academy of Sciences,

Institute of Medicine, Food and

Nutrition Board, Nutrition during

pregnancy, Nutrition during

pregnancy Part I- weight gain. Part

II- Nutrition supplementation,

Committee on Nutritional status

during pregnancy and lactation,

Subcommittee on dietary dietary

intake and nutrient supplements

during pregnancy.1990.

Washington, D C: National

Academy Press.

22. Chin J, Murtaugh M. Socio-

demographic and behavioral

predictors of gestational weight

gain. Am J Obstet Gynecol 2012;

206: S255-S256.

23. Hammad SM, El-Gilany A. The

effect of body mass index on

pregnancy outcomes. The Egyptian

Journal of Community Medicine

2008; 26:15-26.

24. Nisa MU, Aslam M, Ahmed SR,

Rajab M, Kattea L.Impact of

Obesity on Fetomaternal Outcome

in Pregnant Saudi Females. Int J

Health Sci (Qassim) 2009; 3(2):

187–195.

25. Lawoyin TO. Maternal weight and

weight Gain in Africans: Its

relationship to birth weight. J Trop

Pediatr 1991; 37(4):166-171.

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Table 1: Number and percent distribution of studied pregnant women with criteria of

high risk pregnancy

*a pregnant woman may have more than one risk pregnancy factors

Criteria of high risk pregnancy pregnant Women with high

risk of pregnancy factors*

No. %

Obstetric history:

1-Previus stillbirths or neonatal loss 13 4.0

2-History of 3 or more consecutive spontaneous abortions 3 0.9

3-Birth weight of last baby < 2500g 25 7.7

4-Birth weight of last baby > 4500g 2 0.6

5-Last pregnancy hospital admission for H.T. or eclampsia / preeclampsia 3 0.9

6-Previous surgery on reproductive tract 46 14.2

Current pregnancy:

1-Diagnosed or suspected multiple pregnancy 1 0.3

2-Age >40 years 9 2.8

3-Isoimmuization RH(-) in current or previous pregnancy 19 5.9

4-Vaginal bleeding 6 1.9

5-Pelvic mass 2 0.6

6-Blood pressure140/90mmHg or more at booking 2 0.6

7-Cardiac diseases, Diabetes mellitus, or renal diseases 5 1.5

8-Any other severe medical diseases or conditions 75 23.2



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Table 2: Demographic, reproductive characteristics and family history of studied pregnant

women during normal and high risk pregnancies

Characteristics N

Normal

pregnancy

(n=123)

High risk

pregnancy

(n=106)

Test of

Significance

P value 229 No. % No. %

A-Demographic characteristics

1-Age group (years)

15-<25 60 43 71.7 17 28.3

25- <35 137 70 51.1 67 48.9 χ2 =14.651, P<0.01

35- 45 32 10 31.2 22 68.8

2-Nationality

Saudi 217 118 54.4 99 45.6 FET=0.74, P>0.05

Non-Saudi 12 5 4.7 7 57.3

3-Women occupation

House wife 221 121 58.8 100 45.2 χ2 =2.75, P>0.05

Working for cash 8 2 25.0 6 75.0

B- Reproductive characteristics

1-Abortions 59 28 47.5 31 52.5 FET=1.25, P>0.05

2-Stillbirths 2 0 0.0 2 100.0 --------

3-Number of living children

1-2 74 38 51.4 36 48.6 χ2 =17.56, P<0.01

3-4 56 28 50.0 28 50.0

≥5 32 9 28.1 23 71.9

No living children 67 48 71.6 19 28.4

C-Family History

Diabetes Mellitus 121 63 52.1 58 47.9

Multiple pregnancy 29 12 41.4 17 58.6 χ2 =2.899, P>0.05

hypertension 109 61 56.0 48 44.0

Blood disorders 6 4 66.7 2 33.3

Consanguinity 20 9 45.0 11 55.0



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Table 3: Maternal body gain and socio-demographic characteristics of studied pregnant

women during normal and high risk pregnancies

*P<0.05

Table 4: Logistic regression analysis of significant factors predicting maternal body weight gain

among studied pregnant women during normal pregnancy

Maternal Body Gain

Normal pregnancy (n=123) High risk pregnancy (n=106)

Variables Normal

weight gain

Low

weight gain

Over

weight

gain

Normal

weight

gain

Low

weight gain

Over

weight

gain

(8.1-<16) (<8) (≥16) (8.1-<16) (<8) (≥16)

(n=43 ) (n=78 ) (n=2 ) (n=20) (n=85 ) (n=1 )

Age (x ±SD) 25.3±3.9 27.1±5.6 25.0±11.3 29.0 ±5.5 29.8±5.9 -----

Parity (x ±SD) 2.2 ±1.4 3.5 ±2.3* 2.5 ±2.1 3.5 ±2. 9 2.8 ±0.85* -----

Number of Antenatal

care visits (x ±SD) 3.4 ±0.7 3.1 ±0.9 3.0 ±0.001 3.5 ±0. 7 4.5±2.8 -------

Number of living

children (x ±SD) 0.97 ±1.2 1.9±1.8* 2.5±2.1* 2.1 ±1.9 1.9±1.8 --------

Variables B coefficient B P-value O.R.

95 % Confidence

interval of O.R.

Lower Upper

Mother education 0.503 0.2 0.012 1.7 1.12 2.45

Constant 91.19 7.84 ------ ---- ------ -----

Model X2 = 19.95, P >0.05



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Figure 1: Percentage of pregnant women achieved body weight gain during normal and

high risk pregnancy

0

10

20

30

40

50

60

70

80

90

Low weight gain Normal weightgain

high weight gain

63.4

35

1.6

80.2

18.9

0.9

Per

cen

tag

e

Normal pregnancy



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Determinants of Overweight and Obesity among Qatari Children (2-5

years) in Doha, Qatar-2010

Mervat Rady

1, 2*, Mariam Al-Muslemani

1, Rasha Salama

1, 3

1Joint Program of Arab Board in Community and Family Physicians, Doha, Qatar.

2Department of

Community, Environmental and Occupational Medicine, Faculty of Medicine, Ain-Shams University,

Egypt. 3Department of Community Medicine, Faculty of Medicine, Suez Canal University, Egypt

*Corresponding Author: Dr. Mervat Rady, Consultant Trainer -Arab Board Program, Primary Health

Care-Doha, Department of Family and Community Medicine, Qatar. Email: [email protected]

ABSTRACT

Background: Childhood obesity is a major global burden with modifiable and preventable

underlying risk factors. Objective: to determine risk factors for overweight and obesity

among children; 2-5 years old in Doha city, Qatar. Subjects and Methods: A cross sectional

approach was conducted from July 2009 to April 2010. Six primary health care centers were

randomly selected using the cluster sample technique. Data was collected by Pre-designed

and pre-tested interview questionnaire with children's mothers to elicit the information about

children socioeconomic characteristics, dietary history and physical activity. Weight and

height were measured and BMI was calculated. Overweight and obesity were assessed by

WHO- BMI for age growth charts. Results: the overall prevalence of overweight and obesity

was 26.9%. The final model of multiple logistic regression analysis showed that important

determinants of overweight and obesity were positive family history of obesity, low levels of

physical activity and consuming high caloric food items more than 4 times per week.

Conclusion: The study documented high prevalence of overweight and obesity among

preschool children in Doha city. Family history of obesity, low levels of playing outdoors and

dietary pattern were determinants of overweight/obesity. Suitable recommendations were

issued to prevent childhood overweight and obesity among preschoolers.

Key words: Preschoolers, Qatar, Overweight, Obesity, determinants.

Rady M, Al-Muslemani M, Salama R. Determinants of Overweight and Obesity among Qatari

Children (2-5 years) in Doha, Qatar-2010. Canad J Clin Nutr 2013; 1(1): 16-26.

INTRODUCTION

Childhood obesity is a condition where

excess body fat negatively affects a child's

health or wellbeing (1). Developing

countries have seen an increase in child

obesity since they started to adapt the

westernized lifestyles and behaviors with

excess positive energy balance, accelerated

by an increasingly sedentary lifestyle in

recent decades (2, 3). The increasing

consumption of fats, sweeteners, energy

dense foods, and fast food meals compared

to traditional diets with higher intake of

cereals characterize the current era. The

result is a series of nutritional inequities in

many nations, communities, and

households. Technology and transportation

throughout the food supply chain have

facilitated the production and distribution

of foods associated with the nutrition

transition, while cultural influences have

introduced new foods into developing

countries and shaped the desire for those

foods (4, 5). State of Qatar is being

classified among the countries passed

through advanced nutritional transition

stage, with high level of overweight and

Page 16-26




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obesity, and micronutrient deficiencies in

some population sub-groups (6).

Determining the risk factors of preschool

overweight and obesity can serve as a

basis for planning policies and cost

effective intervention programs as well as

a baseline for further research. So the aim

of this study was to identify determinants

of overweight and obesity among

preschool children aged 2 to 5 years in

Doha city.

SUJECTS AND METHODS

Study Design

This cross-sectional study was conducted

from July 2009 to April 2010 in sample

units of six randomly selected primary

health care centers (PHCCs) from the total

10 centers list of Al-Doha city; the capital

of Qatar.

Sample Size

The sample size was estimated by using

the formula: n= DEFFECT [z2 α/2 (pq)

/d2]; the prevalence of obesity was taken

as 16% (7), the degree of precision of the

estimate was set at 5% and the design

effect of cluster sampling was set as 1.8.

Using the previous formula the sample

size was estimated to be 371. After adding

an inflation rate 30%, an additional 112

children were added. Thus 483 children

were selected for this study.

Study Population

The study subjects were apparently healthy

children including boys and girls aged 2-5

years who attended these centers with their

mothers and agreed to participate by

maternal written consent. The following

children were excluded; children with

chronic diseases or using medications

capable to interfere with weights or

heights, and children with physical

deformities or if the mothers were

unwilling to participate. The study

protocol was approved by the ethical

committee of Hamad Medical Corporation

and Primary Health Care Corporation. A

prior consent for the study was taken from

the mothers.

Study Tools

At each center, a structured interview

questionnaire was used. It consists of 36

closed ended questions in Arabic about:

Children‗s characteristics: age, sex, child

siblings‗ number and birth order; parents

characteristics: educational level, maternal

job, family income and family history of

obesity; children‗s behaviors and habits as

regard eating habits, daily activity and

exercise and the recorded anthropometric

measurements of the child and his/her

mother. Data collection process was done

separately in a private room for each child

to ensure privacy. The pretest of the

questionnaire had been done prior to the

real data collection among 30 mothers.

Weights and heights were taken by trained

nurses in the centers using the standard

techniques with a sensitivity of 0.1 cm and

0.1 kg respectively. Zero error was set

daily. BMI was calculated and the

children were identified as overweight if

the BMI was ≥85th- <97th percentile and

obese if BMI was ≥97th percentile

according to age-sex specific percentiles of

BMI using the WHO BMI-for-age growth

charts (8).

Exclusive breast feeding: was defined as

that the child being fed no food or drinks

even the water other than the breast milk.

Eating behaviors and habits: they were

based on food frequency. It is concerning

type of food usually consumed by Qatari

children in a week. The categories of each

food item were classified by the author as

follow: > 4 days per week: Every day or 5-

6 times per week, ≤ 4 times per week:

equal to and below 4 times per week and

Never/very seldom: never or 2 days or 1

day per month. Daily life activity: This

refers to the average length of time

children spend on each activity in a day.

The reference for watching television, and

playing indoor games (such as computer

games, game boy, and play station) were

considered to be < 2 hours per day. For



Pag

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playing outdoors, we considered the

reference to be ≥ 1 hour by day. Exercise:

This refers to whether the child

participates in any type of sports, or

exercise suitable for his/her age; the ideal

frequency per week was considered 3

times per week and duration per time not

less than 30 minutes.

Statistical Analysis

All data entries were double checked for

any errors. Association of each categorical

variable with overweight and obesity was

assessed using X2 test and the strength of

their association was computed by the

unadjusted odds (95% confidence

interval). Variables showing statistically

significant (P<0.05) association with the

outcome variable (overweight and obesity)

were considered as risk factors for the

outcome. These variables were

simultaneously entered into a step wise

logistic regression model to determine the

independent risk factors of overweight and

obesity. Data analysis was performed

using the SPSS statistical package. In this

study P value < 5 % was considered as

statistical significant.

RESULTS

A total 464 children from the selected 6

PHCCs who satisfied the inclusion criteria

and agree to participate, were enrolled in

the study, out of them 50.2% were boys

and 49.8 were girls with mean sample age

41.69 ±10.2 months. 98 of the studied

children were under weights according to

body category and excluded from the

analysis. The overall prevalence of

overweight and obesity was 12.9% and

14%. The prevalence of overweight and

obesity was 10.6% and 15.5% in boys and

15.2 and 12.5% in girls. The overall

difference between boys and girls was

statistically insignificant.

Table 1 represents the bivariate

relationship of some socioeconomic and

nutritional habits variables were

statistically significant (P<0.05) and were

associated with increased risk of

overweight and obesity as the odds ratios

exceeded the unity, while for high protein

and green vegetables intake; both were

significant protective variables and the

since the odds ratio was 0.05 and 0.41

respectively.

Table 2 shows the statistically significant

bivariate relationship of some physical

activity variables which were associated

with increased risk of overweight and

obesity as the odds ratios exceeded (1) and

the 95% CI does not contain the unity,

P<0.05. The data on table 2, also shows

that there is a very low percent 11.2%

(41/366) of children who practice sports or

exercise in our sample and there is no

relation between the history of doing

exercise and occurrence of OW&OB

among the children since P>0.05.

However, the duration and frequency per

week were significantly associated with

occurrence of OW&OB among children in

this age group since higher percent of

OW&OB were among those practice

sports for less than 30 minutes per time

and less than 3 times per week compared

to the other categories and P< 0.001 in the

two situations.

Table 3 illustrates that, when the variables

showing significant association at P<0.05

were simultaneously considered in the

logistic regression model with overweight

and obesity as a binary outcome versus the

normal weights, it was observed that

children with positive family history of

obesity, negative history of daily breakfast

and with exclusive breast feeding ≤ 4

months were having 7.4 times (OR= 7.4;

95% CI =2.9-12.8), 7.9 times (OR=7.9;

95% CI=4.1-19.7) and 8.5 times (OR=8.5;

95% CI=5.7-18.3) more risk of

overweight and obesity respectively.

Similarly, children eating meals full of

carbohydrates, bakery and sugary foods

for more than 4 days per week having 8.5

times (CI=1.6-12.5), 3.7times (CI=1.5-9.3)

and 6.2 (4.1-15.5) times more risk of

overweight and obesity respectively. The



Pag

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risk of overweight and obesity is nearly

twice when the child consumed fast food

meals more than 4 times/week (OR=1.9,

CI=1.2-4.1). Playing outdoors in the

weekends for less than 1 hrs/day was

associated with the risk of overweight and

obesity of 18.9 times (OR=18.9; CI=11.3-

26.7).

DISCUSSION

In the present study, the prevalence of

overweight and obesity was 12.9% and

14%; being 10.7% and 15.5% in boys and

15.2% and 12.5% in girls, respectively. In

a study done by Supreme council of Health

in coordination with the WHO-EMRO

(2008), the prevalence of overweight

among less than 5 years Qatari children

was 28.7% (6). Also, Al-Najeeb et al.,

showed that the rate of obesity and

overweight was 23% and 16% respectively

among primary school Qatari children (9).

The results of these studies mean that the

problem of overweight and obesity among

the Qatari children is a real health problem

and reflecting the negligence or

underestimation of the problem. Although

the mechanism of obesity development is

not fully understood, it is confirmed that

obesity occurs when energy intake exceeds

energy expenditure. There are multiple

etiologies for this imbalance, hence, and

rising prevalence of obesity cannot be

addressed by a single etiology. Genetic

factors influence the susceptibility of a

given child to an obesity-conductive

environment. However, environmental

factors, life style preferences, and cultural

environment seem to play major roles in

the rising prevalence of obesity worldwide

(10). Parental obesity is the best predictor

of childhood obesity as reported by Abdul

Wahab et al., in Kuwaiti (11). Also,

Shepherd (2009), reported that the first

cause of excess body fat in children is a

genetic factor especially among near

relatives (12). From the family history of

obesity point of view, the present study

shows strong relation between family

history and children body status suggesting

the genetic effect. This relation may be

due the fact that parents and children

usually share dietary habits and have

similar physical activity levels, thus

contributing towards the greater risk of

obesity in certain families. Also, busy

families today rely on convenient foods

which often come from fast food

establishments, other restaurants, and the

frozen and prepackaged food sections of

the grocery store.

Hurried families no longer have time to sit

down to eat meals together, even though

eating together has been associated with

greater intake of foods from the basic food

groups in both children and adults,

furthermore it was reported that infant

weight gain is associated with an

interaction between the duration of

breastfeeding and the timing of

complementary food introduction (13).

Moreover, they specify the interaction

which was identified for longer durations

of breastfeeding than 20 weeks. In the

meantime, they recognize earlier

complementary food induction (< 16

weeks) was associated with greater infant

weight gain. In a wide-ranging meta-

analysis, Harder et al., included 17 studies,

reported the duration of breast feeding;

and included children fed exclusively with

infant formula for comparison (14). Based

on these data, they reported that the

duration of breast feeding was inversely

associated with risk of overweight,

independent of the definition of

overweight used and the age of the

participants in the studies.

Fast food intake is one of the risk factors

for obesity and overweight in the present

study. It is more energy dense and higher

in fat content. The same result was

observed by David et al and Weyermann et

al., as the prevalence of overweight and

obesity grew from in children with

frequent snaking and consumption of junk

food (15, 16). Excess consumption of



Pag

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carbohydrates, bakery, fatty and the sugary

items were significant risk factors for

overweight and obesity. The same result

was obtained in a number of cross-

sectional and longitudinal studies (10, 17,

18). Consumption of these items increases

weight as they add more calories and

satiety is not addressed. However, Food

frequency methods like the one we used in

our study, measure usual diet, but estimate

caloric intake poorly (10). Other methods

such as 24-hour recall or food diaries

evaluate caloric intakes more accurately,

however, estimate short-term not long-

term intake (19). Mahshid et al., reported

that, total energy intake is difficult to

measure accurately at a population level

(10).

A number of studies in western countries

failed to correlate the excess in obesity

rates with the total energy intake and even

they reported lower level of energy intake

among obese children compared with their

lean counterparts (20). Evaluation of food

intake among pre-school children is an

even more difficult task because there is

little data about food habits and

preferences of this age group in the

literature. Older age groups especially

schoolchildren, teens and adolescents

have been investigated much more

thoroughly (21). Also, food evaluation

using parents recall is always liable to bias

due to over or under estimation as reported

also by Schonfeld and Warden, who

reported that this method is not a valid

measurement (22). Our results document

the relation between missing breakfast and

occurrence of overweight and obesity

among children. The odds to become an

overweight or obese child are 8 times more

among children skipping breakfast than the

children do not do that. Unfortunately,

28% of study sample skips to take the

breakfast daily. It was reported that one

out of every three children skips breakfast.

Skipping breakfast expose a hungry child

to eat more portions from the high caloric

lunch meal or to snack frequency (9).

The role of physical inactivity in the

process of obesity is very clear in the

present study as the obesity rate is

significantly higher among the inactive

group of children. It has been hypothesized

that a steady decline in physical activity

among all age groups has heavily

contributed to rising rates of obesity all

around the world. Numerous studies have

shown that sedentary behaviors like

watching TV and playing computer games

are associated with increased prevalence of

obesity (23). Furthermore, parents from

these studies report that they prefer having

their children watch TV at home rather

than play outside unattended because

parents are then able to complete their

chores while keeping an eye on their

children (24).

In addition, increased proportions of

children who are being driven and low

participation rates in sports particularly

among female children (25), are also

associated with increased obesity

prevalence which is very obvious among

our sample since a very small percent of

the children participate in exercise or sport

activity. Since both parental and children‘s

choices fashion these behaviors, it is not

surprising that overweight children tend to

have overweight parents and are

themselves more likely to grow into

overweight adults than normal weight

children (26). Although, the population-

based studies which relate physical activity

to obesity are limited, but they concluded

that increased physical activity might

decrease the accelerated weight gain

epidemic (27-30). In the present study, it

was found that children who did not play

outdoor games had an increased risk of

overweight and obesity. By encouraging

children to play outdoor games not only

increases their physical activity but also

they remain fit.

CONCLUSION

The major conclusion drawn from this

study is that family history of obesity,



Pag

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absence of daily breakfast, consuming fast

food, excess carbohydrates, bakery and

sugary food items with low levels of

physical activity are associated with higher

prevalence of overweight and obesity

among Qatari preschool children attending

the primary care centers. Accordingly,

there is a need to apply nutritional

education programs to mothers regarding

feeding of their children during this age

group (2-5 years), and encouraging out-

door activities suitable for their age as a

preventive strategies for childhood

overweight and obesity.




study.

Authors' Contribution





manuscript.

Study Limitations

There were certain limitations of the

present study; the study relied on the

mother's memory-recall which influenced

by limited degree of loss of information as

regard their children physical activity and

dietary patterns. Dietary history was based

only on qualitative data and didn‘t take

into consideration type and amount of

foods in details.

Acknowledgements

We would like to thank all nurses,

technicians and physicians for their kind

support in data collection and laboratory

analysis. We also thank the children and

their parents for contribution in the study.

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Table 1: Bivariate relationship between the socioeconomic and dietary habit variables,

overweight and obesity among Qatari children 2-5 years old attending PHCCs-2010

Variables

Body Category

Overweight

& Obese

(n=125)

No. (%)

Normal

(n=241)

No. (%)

OR (95%CI)

P-Value

Positive family history of obesity 80 (64.0) 28 (11.6) 13.2 (7.5-23.5) <0.001

Exclusive Breast feeding ≤ 4 months 114 (91.2) 189 (78.4) 2.9 (1.4-5.7) <0.001

> 3 meals per day 40 (32.0) 40 (16.6) 2.4 (1.4-4.1) <0.001

Negative history of daily breakfast 80 (64.0) 34 (14.1) 10.8 (6.3-18.7) <0.001

Snacks consumption ≥ onetime/day 92 (73.6) 142 (58.9)

1.94 (0.8-4.5) <0.05

Soft drinks consumption ≥ onetime/day 97 (77.6)

137 (56.8)

2.74 (1.5-5.1)

<0.001

Artificial juice consumption ≥ onetime/day 105 (84)

121(50.2)

17.4 (4.1-23.6)

<0.001

Protein intake > 4 days/week 16 (12.8)

57 (23.7)

0.05 (0.01-0.4) <0.001

Dairy products intake > 4 days/week 69 (55.2) 96 (39.8) 1.86 (1.2-2.9) <0.01

Carbohydrate products intake > 4 days/week 34 (27.2)

8 (3.3)

36.(8.9-150.7) <0.001

Sugary products intake > 4 days/week 76 (60.8)

15 (6.2)

14.8 (6.3-34.9)

<0.001

Bakery products intake > 4 days/week 64 (51.2)

18 (7.5)

5.5 (2.2-13.8) <0.001

Fatty meals intake > 4 days/week 6 (4.8) 2 (0.8) 11.1 (2.1-59.2) <0.001

Fast food meals intake > 4 days/week 24 (19.2)

23 (9.5)

3.6 (1.7-7.9) <0.001

Green vegetable intake ≥ onetime/day 78 (62.4)

173 (71.8)

0.41(0.17-0.98)

<0.05

Fruits & fresh juice intake ≥ onetime/day 18 (14.4)

12 (4.9)

2.9 (1.3-6-7)

<0.01



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Table 2: Bivariate relationship between the physical activity habits variables,

overweight and obesity among Qatari children 2-5 years old attending PHCCs-2010

Variables

Body category

Overweight &

Obese(n=125)

N0. (%)

Normal

(n=241)

No. (%)

OR (95%CI) P-Value

Spend time for sleeping > 8 hrs/day 57 (45.6) 46 (19.1) 3.55 (2.2-5.9) <0.001

Spend time watching TV > 2 hrs/day 103 (82.4) 166 (68.9) 2.12 (1.2-5.9) <0.01

Spend time playing other electronic

games > 2 hrs/day

33 (26.4) 21 (8.7) 4.32 (2.4-7.9) <0.001

Spend time playing outdoors in

weekdays < 1 hr/day

90 (72) 124 (51.4) 2.43 (1.5-3.9) <0.001

Spend time playing outdoors in

weekends < 1 hr/day

114 (91.2) 37 (15,4) 57.1(27.1-67.2) <0.001

Positive history of practicing exercise 18 (14.4) 23 (9.5) 0.63(0.31-.29) >0.05

Time sport <30 min/time 18 (72.2) 23 (13.04) 17.3 (3.5-85.2) <0.001

Frequency/week <3 times/week 18 (88.8) 23 (26.1) 22.7(3.9-29.1) <0.001



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Table 3: Risk factors using stepwise logistic regression analysis

Variables Category AOR (95%CI) P-Value

Family history of obesity No

Yes

1.00

7.4 (2.9-12.7)

<0.01

Exclusive breast feeding > 4 months

≤ 4 months

1.00

8.5 (5.7-18.3)

<0.01

Daily breakfast intake Yes

No

1.00

7.9 (4.1-19.7)

<0.001

Meals full of carbohydrates ≤ 4 days/week

> 4 days/week

1.00

8.5 (1.6-12.5)

<0.05

Bakery foodstuffs intake ≤ 4 days/week

> 4 days/week

1.00

3.7 (1.5-9.3)

<0.001

Sugary foodstuffs intake ≤ 4 days/week

> 4 days/week

1.00

6.2 (4.02-15.5)

<0.001

Fast food meals intake ≤ 4 days/week

> 4 days/week

1.00

1.9 (1.2-4.1)

<0.001

Playing outdoors in weekends ≥ 1 hr/day

< 1 hr/day

1.00

18.9 (11.3-26.7)

<0.001

Constant -57.023

The overall predictive % of the model=94% (R2

= 0.943)



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Anemia among Primary School Children (5 -12 years) in Riyadh Region,

Saudi Arabia: A Community-Based Study

Ashry Gad

1*, Joharah AL-Quaiz

1, Tawfik Khoja

2, Abdullah As-Sharif

3, Hamad Al-Manea

3,

Abdullah Al Edriss3, Sheikh Shafi

1

1Department of Family and Community Medicine, College of Medicine, King Saud

University, 2 Director Executive Board Council for Gulf cooperation States, 3Ministry of

Health, Riyadh, Saudi Arabia.

*Corresponding Author: Prof. Ashry Gad Mohamed. Email: [email protected]

ABSTRACT

Background: Anemia among school aged children is known to be an important global

public health problem in both developing and developed countries. It affects the physical and

intellectual functions of the affected children. School years are ideal opportune time to

intervene to prevent and control anemia. Objectives: The objective of the study is to

investigate the frequency of anemia and the associated dietary and medical risk factors in

school aged children in Riyadh region. Subject and Methods: A cross sectional survey was

carried out in Riyadh region. The study sample was selected using the two stages of cluster

sampling technique. Standardized Arabic questionnaire was completed by parents of school

aged children by two well trained nurses. Dietary frequency was requested for the last week

prior to the interview. A venous blood sample was taken for hemoglobin estimation. Anemia

in school aged children was defined according to the WHO definition. Results: The total

sample was 1117 children, 49.9% males and 50.1% females. Prevalence of anemia was

22.3% (22.4% in males & 22.2% in females). Frequent eating of red meat reduced the risk of

anemia (OR=0.8). Frequent drinking of cola or sour milk (Laban) with lunch meal

significantly increased the risk of anemia (OR=1.52, 1.06-2.16 and OR=1.55, 1.07-2.25

respectively). Family history of hereditary blood disorders or iron deficiency anemia

increased the risk of anemia in school aged children (OR=5.48, 1.02-31.21 and OR= 3.38,

1.74-6.54 respectively). Conclusions: Anemia in school children is a moderate public health

problem in Riyadh region. Drinking sour milk with lunch and positive family history

increases the risk of anemia in school children.

Key words: anemia, school aged children, Diet.

Gad A, AL-Quaiz J, Khoja T, As-Sharif A, Al-Manea H, Al-Edriss A, Shafi S. Anemia among

primary school children (5 -12 years) in Riyadh region, Saudi Arabia: a community-based

study. Canad J Clin Nutr 2013; 1(1): 27-34.

INTRODUCTION

Anemia prevalence rate in pediatric

population can be an important indicator

of nutritional status (1).

Iron deficiency is

the main cause of anemia; therefore

anemia prevalence can be used as a proxy

for prevalence of iron deficiency anemia

(2). Anemia among school-aged children is

known to be a significant global public

health problem affecting 305 million

people around the world. In developing

countries its prevalence ranges from

29.2% to 79.6% (3). Even in USA as many

as 20% of children are assumed to become

anemic at some point by the age of 18

years (4). It can cause many adverse

effects on child health, including delayed

psychomotor development, impaired

cognitive function, IQ loss and increased

Page 27-34




Pag

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susceptibility to lead toxicity (5, 6).

Correction of iron deficiency at this age

group enhances children‘s learning

potential in schools, increases their fitness

and work capacity. In addition

improvement of girls‘ iron status may help

to prevent anemia during the reproductive

years (7-10).

The prevalence of anemia as a public

health problem is categorized as follows:

<5% no public health problem; 5-19.9%

mild public health problem; 20-39.9%

moderate public health problem and ≥40%

severe public health problem (8-11). Only

two countries among 192 WHO member

states anemia is not a public health

problem. It is mild in 40, moderate in 81

and severe in 69 states (11). The school

years are an opportune time to intervene,

and indeed the school setting offers an

ideal distribution system, and interventions

must be based on sound epidemiological

understanding of the problem at this age

group (11). Nutritive status of school aged

children is vulnerable (3); we therefore

conducted this cross sectional survey to

investigate the frequency of anemia and

the associated dietary and medical risk

factors in school aged children in Riyadh.

SUJECTS AND METHODS

Study design and setting

A Community based household cross

sectional survey was conducted over a

period of 4 months. Saudi subjects from

Riyadh region, older than one year, were

recruited on voluntary basis as a reference

population for this study. Riyadh region,

with a population amounted to 3.726.523

Saudi persons (Ministry of Planning,

preliminary results of 1425 (2004 G)

census. The study was approved by

Ministry of Health.

Sampling

The sample size was calculated on the

assumption of 30% prevalence of IDA in

Riyadh (5). At 95% confidence level and

acceptance of 2% as degree of precision,

the sample size was 2016 persons if the

simple random method was used. Due to

the large reference population, cluster

sampling technique was applied and a

design effect of 2 was used. The sample

size increased to 4032. A response rate for

blood extraction was assumed to be 60-

70% and accordingly the final sample size

was calculated to include 6204

participants. Out of these 1117 school

aged children were included in this study.

Two stage cluster sampling technique was

used to accomplish the study. In the first

stage 36 clusters were selected

proportionally allocated with the number

of population in each PHCC catchments

area. In the second stage a constant

number was selected per cluster.

Assuming that the average Saudi family in

Riyadh region is 7 persons, 25 households

were included in each cluster.

Data Collection

A Standardized Arabic questionnaire-

interview was designed to satisfy the study

objectives. It investigated about socio-

demographic characteristics, dietary,

medical history of each child 5-12 years

old within the family. Data were collected

by two well trained nurses. For nutritional

status assessment the dietary frequency

was requested for the last week prior to

interview. A venous blood sample was

taken for hemoglobin estimation. Anemia

was defined according to WHO

hemoglobin level in Children 5-11.99

years (< 11.5 g/dl) (12).

Statistical Analysis

The data was entered and analyzed, using

SPSS PC version 17.0 statistical software.

The outcome variable was dichotomized as

anemia (presence or absence). The study

variables related to socio-demographic,

medical history and dietary assessments

were used to observe and quantify the

statistical association. Odds ratio (OR) was

used to measure an association between



Pag

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the two categorical variables. To adjust

odds ratios of the variables that proved to

be statistically associated with the

occurrence of anemia in the bivariate,

analysis were entered into multivariate

logistic regression model. The 95%

confidence intervals for adjusted odds

ratios were calculated. A p-value of <0.05

was considered as statistically significant.

RESULTS

Total 1117 school aged children were

included for this study. Out of this 557

(49.9%) were males and 560 (50.1%) were

females. Only 753 (67.4%) parents gave

consent for blood sampling. Prevalence of

anemia was 168 (22.3%), with no

statistically significant difference between

males and females (P>0.05).Table 1 shows

that frequent eating of red meat reduced

the risk of anemia (OR=0.8), however this

risk reduction was not statistically

significant (CI=0.56 -1.14). Frequent

drinking of cola or sour milk (Laban) with

lunch meal significantly increased the risk

of anemia (OR=1.52, 1.06-2.16 and

OR=1.55, 1.07-2.25 respectively).

Blood related disorders in the child and/or

his family are shown in Table 2. Family

history of hereditary blood disorders or

iron deficiency anemia increased the risk

of anemia in school aged children

(OR=5.48, 1.02-31.21 and OR= 3.38,

1.74-6.54 respectively). Personal history

of blood transfusion and IDA were risk

factors for anemia in the studied children

(OR=11.48, 1.06-288.4 and OR=3.11,

1.57-6.17 in order).

Table 3 displays results of multivariate

logistic regression analysis and revealed

that drinking sour milk (Laban) with lunch

and family history of anemia were

independent risk factors for developing

anemia in school aged children in Riyadh

region.

DISCUSSION

The present study revealed that anemia is a

moderate public health problem among

school aged children in Riyadh region

(22.3%). A national study conducted

eleven years ago reported prevalence of

anemia of similar figure of 24.8% among

Saudi children younger than 14 years with

the highest prevalence being in the Eastern

region (41.3%) (13). The former study

used hemoglobin level of 11.2 gm/dl as a

threshold for anemia which is lower than

the current WHO (11.5 gm/dl) used in the

current study. Also another study in

Jeddah that included 2000 school children

found that 20.5% of them were anemic

(14). The low prevalence of anemia in

North America may be attributed to the

fortification of iron in most of the

available foods there (15).

Al-Othaimeen et al., conducted a study

among 1210 school girls aged 7-14 years

old in Riyadh city and found that 30% had

hemoglobin level <11 gm/dl. Prevalence

rate of the latter study is higher than the

present work (22.2%) (16), this may be

attributed to the inclusion of girls aged 12-

14 years where most of them were

menstruating. Higher rate was also

reported by Rasheed et al., who

investigated 285 primary school girls in

urban area of Al-Khober in the Eastern

province of Saudi Arabia. They found that

26.4% of girls had anemia (hemoglobin

level <11 gm/dl) (17).

Haemoglobin level was used in this study

as an indicator for Iron status in the body.

Kihli-Kumar recommended the assessment

of hemoglobin as the most common

method of assessing anemia (3, 18).

School children were chosen for the study

as rapid growth in school aged children

increases the demand for iron (19) and

anemia is estimated to affect one half of

school age children in developing

countries (20). The school years are an

opportune time to intervene, and

interventions must be based on sound

epidemiologic understanding of the

problem in this age group (2). It has been

suggested that when anemia prevalence is

20%, iron deficiency exists in 50% of the



Pag

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population and when anemia prevalence is

greater than 40% the entire population

suffers from some degree of iron

deficiency.

Several dietary factors can influence iron

absorption. Enhancing factors are ascorbic

acid which is found in citric fruits, meat,

fish and poultry. Inhibitory factors include

tannic acid which is found in tea, coffee

and chocolate, and calcium which is

present in milk and dairy products (21,

22). Cross sectional surveys revealed that

the frequency of meat and dairy

consumption determines hemoglobin

values in school age children (23).

In the

present work meat was a protective factor

in bivariate analysis (OR=0.8). Sour milk

called Laban by the Saudi public was

approved to be a risk factor for anemia in

school aged children (OR=1.48). Its high

contents of calcium may explain this

finding particularly as it is used during or

just after lunch meal which is the main

meal of the Saudi population, thus

inhibiting the absorption of iron.

Drinking tea or coffee in the current work

did not influence the prevalence of anemia

(p>0.05). On the same line Temme and

Van Hoydonck conducted a PubMed

search and reported that tea consumption

does not influence iron status in Western

countries in which people have adequate

iron stores. Only in populations with

marginal iron status there seems to be a

negative association between tea

consumption and iron status (24). Poulter

J and Nelson M reviewed 35 references

investigating the impact of drinking tea on

iron status and reported that according to

the available evidence restriction of

drinking tea is advised only to groups at

risk of iron deficiency. It is advised to

drink tea between meals or to wait at least

one hour after eating before drinking tea

(25).

Presence of positive family history of

anemia is an independent risk factor for

occurrence of anemia in school aged

children in the current work (OR=2.42,

1.18-4.94). This may be due to common

exposure to iron deficient diets by the

family or presence of hereditary blood

diseases within the family. In addition, this

diagnosis of IDA in Saudi Arabia is more

complicated with the high incidence of

hemoglobin disorders such as sickle cell

and thalassemia traits (26, 27).

CONCLUSION

Anemia is a moderate public health

problem among school aged children in

Riyadh region. Iron fortification is highly

recommended. Health education programs

should be conducted in schools to

highlight the risk factors of anemia

particularly drinking sour milk with meals.

Children with family history of anemia

should be screened for anemia and treated

if indicated.




study.






manuscript.

Limitation of the Study

The limitations of the present study are the

relatively low response rate due to fear of

children and their parents from blood

extraction, and the lack of classification of

nutritional anemia into IDA and non-iron

deficiency anemia as ferritin level

estimation was not done.

Acknowledgements

We would like to thank all nurses,

technicians and physicians for their kind

support in data collection and laboratory

analysis. We also thank the children and

their parents for contribution in the study.

This study was funded by Saudi Ministry

of Health.



Pag

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1. Oken E, Rifas-Shiman SL, Kleinman

KP, Scanlon KS, Edwards JWR, Gillman

MW. Trends in childhood anemia in a

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2. Dallman PR, Yip R, Johnson C.

Prevalence and causes of anemia in the

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Radovic LC, Rakic L, Kocic S, Davidovic

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among Serbian school age children 7-14

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4. Irwin JJ, Kirchner JT. Anemia in

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5. Lozoff B, De Andraca I, Castillo M,

Smith JB, Walter T, Pino P. Behavioral

and developmental effects of preventing

iron-deficiency anemia in healthy full term

infants. Pediatrics 2003;112: 846-854.

6. Grantham-Mcregor S, Ani C. A review

of studies on the effect of iron deficiency

on cognitive development in children. J

nutr 2001;131:649S-666S.

7. DeMaeyer EM, Adiels-Tegman M. The

prevalence of anemia in the world. Rapp

Trimest Stat Sanit Mond 1985;38:302-3l6.

8. Ham ID, Fairchild MW. Summary and

conclusions of the International

Conference on Iron Deficiency and

Behavioral Development. Am J Clin Nutr

l989;50:703-705.

9. Stephenson LS, Latham MC, Adams El,

Kinoti SN, Pertet A. Physical fitness,

growth and appetite of Kenyan school

boys with hookworm, Trichuris trichiura

and Ascaris lumbricoides infections are

improved four months after a single dose

of Albendazole. J Nutr 1993;123: 1036-

1046.

10. Savioli L, Bundy DAP, Tomkins A.

Intestinal parasitic infections: a soluble

public health problem. Trans R Soc Trop

Med Hyg 1992; 86:353-359.

11. Maclean M, Egli CI, Wajdyla D,

Benoist B. Worldwide prevalence of

anemia. In: Nutritional anemia. Edited by

Kraemer K & Zimmermann MB. Sight and

Life Press. pp: 3- 12., 1st, 2007.

12. Benoist BD, Mclean E, Egli I,

Cogswell M. Worldwide prevalence of

anemia 1993-2005. WHO 2008. Spain.

13. El-Hazmi MA, Warsy AS. The pattern

of common anemia among Saudi children.

J Trop Pediatric 1999; 45(4):221-225.

14. Abalkhail B, Shawky S. Prevalence of

daily breakfast intake, iron deficiency

anemia and awareness of being anemic

among Saudi school students. Int J Food

Sci Nutr 2002; 53(6): 519-528.

15. Hurrell RF, Jacob S. Role of the food

industry in iron nutrition: iron intake from

industrial food products. In: Hallberg L,

Asp NG, eds. Iron nutrition in health and

disease. London: John Libbey & Co., 1996

pp. 339–347.

16. Al-Othaimeen A, Osman AK, Al-Orf

S. Prevalence of nutritional anemia among

school girls in Riyadh city, Saudi Arabia.

Int J Food Sc Nutr 1999;50:237-243.

17. Rasheed P, Al-Yousef N, Al-Dabal B.

Nutritional profile of Saudi primary school



Pag

e32

girls in an urban region. Ann Saudi Med

1989;9:371-377.

18. Kihli-Kumar M. Screening for anemia

in children: AAP recommendations: a

critique. Pediatrics 2001;108:1-2.

19. Brabin L, Brabin BJ. The cost of

successful adolescent growth and

development in girls in relation to iron and

vitamin A status. Am J Clin Nutr 1992;

55:955-958.

20. Stoltzfus RJ, Chwaya HM, Tielsch JM,

Schulze KJ, Albonico M, Savioli L.

Epidemiology of iron deficiency anemia in

Zanzibari schoolchildren: the importance

of hookworms. Am J Clin Nutr

1997;65(1):153-159.

21. Zijp IM, Korver O, Tijburg LB. Effect

of tea and other dietary factors on iron

absorption. Crit Rev Food Sci Nutr

2000;40(5): 371-398.

22. Coutinho GGPL, Bertollo EMG,

Bertelli ECP. Iron deficiency anemia in

children: a challenge for public health and

for society. Sao Paulo Med J 2005;

123(2):11-22.

23. Khatib IM, Elmadf I. Poor nutritional

health of Bedouin preschool children in

Jordon: the irony of urbanization. Ann

Nutr Metab 2009; 54:301-309.

24. Temme Eh, Van Hoydonck PG. Tea

consumption and iron status. Eur J Clin

Nutr 2007; 56(5): 379-386.

25. Nelson M, Poulter J. Impact of tea

drinking on iron status in the Uk: a review.

J Hum Nutr Diet 2004; 17(1):43-54.

26. Al-Hazmi MA, Jabbar FA, Al-Faleh

FZ, et al. Patterns of sickle cell

thalassemia and glucose-6 phosphate

dehydrogenase deficiency gene in north-

western Saudi Arabia. Hum Hered

1991;4:26-34.

27. Warsy AS, El-Hazmi MA. Glucose-6

phosphate dehydrogenase deficiency in

Saudi Arabia- a review. Saudi Med J

1987;8:12-20.



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Table 1: Dietary factors and anemia in school children, Riyadh, Saudi Arabia

Food Intake Status Odds

Ratio

95% Conf.

Interval

P

Anemic healthy

Frequent eating of

red meat

Yes

No

93

83

341

243

0.8 0.56 -1.14 0.19

Frequent eating of

vegetable

Yes

No

115

60

358

223

1.19 0.83 – 1.73 0.33

Frequent drinking

of Tea & coffee

Yes

No

34

141

145

435

0.72 0.47 – 1.12 0.13

Frequent drinking

of Cola

Yes

No

86

86

229

347

1.52 1.06 – 2.16 0.017

Frequent drinking

of sour milk

(Laban) with

lunch

Yes

No

115

57

328

252

1.55 1.07 – 2.25 0.016

Frequent drinking

of juice

Yes

No

49

123

133

442

1.32 0.89 – 1.98 0.15



Pag

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Table 2: Blood related disorders and anemia in school children, Riyadh, Saudi Arabia

Health

problem

presence Status Odds

Ratio

95% Conf.

Interval

P

Anemic Healthy

Personal

history of

hereditary

blood

disorder

Yes

No

8

143

14

558

2.23 0.84 – 5.8 0.10

Family

history of

hereditary

blood

disorder

Yes

No

4

128

3

526

5.48 1.02 – 31.21 0.01

History of

blood

transfusion

Yes

No

3

151

1

578

11.48 1.06 – 288.4 0.008

Current

blood

disorder

Yes

No

6

156

2

592

11.38 2.00 – 115.9 0.002

Past history

of IDA

Yes

No

18

131

24

544

3.11 1.57 – 6.17 0.0005

Family

history of

IDA

Yes

No

20

129

25

545

3.38 1.74 – 6.54 0.00005

Table 3: Results of logistic regression analysis

Variable Adjusted

Odds Ratio

95% Conf. Interval P

Drinking cola with lunch 1.36 0.94 – 1.99 0.108

Drinking sour milk ( laban)

with lunch

1.48 1.00 - 2.19 0.050

Family history of anemia 2.42 1.18 – 4.94 0.015

NB. The model explains 79.2% of occurrence of anemia.



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Dietary Practice and Physical Activity in Children with Down Syndrome

and Their Siblings in Saudi Arabia

Badreldin A Mohamed1*

, Adel A Alhamdan1, Manal M Samarkandy

2

1Department of Community Health Sciences, College of Applied Medical Sciences, King

Saud University, Riyadh, Saudi Arabia. 2 Manal M. Samarkandy, Senior Dietitian, Sultan Bin

Abdulaziz Humanitarian City, Riyadh, Kingdom of Saudi Arabia.

*Corresponding Author: Dr. Badreldin A Mohamed. Email: [email protected]

ABSTRACT

Background: Feeding difficulties and inappropriate nutrition are common problems among

children with Down Syndrome (DS). Objective: The aim of this study is to investigate the

dietary practice and physical activity among children with DS. Methodology: The study

groups were pre-pubertal DS boys and girls, aged 5 to 12 years clinically and cytogenetically

proven to be suffering from DS. Healthy siblings, closest in age to the DS children were used

as a control group. Breast feeding, eating difficulties, fast food intake, and physical activity

were measured for both groups. Results: During infancy period, 36.4% of the DS children

were bottle fed, compared to only 5.5% of the normal siblings. Nearly half of the breast-fed

DS children were fed for duration of less than 6 months. The percentage of the DS children

experiencing dietary difficulties is significantly higher compared to the siblings. Concerning

physical activity, 73.1% of DS children did not exercise as compared to 44.2% of the control

siblings. Conclusion: Controlling feeding practices and encouraging Down syndrome

children to participate in physical activity either through support from parents or through

designing special programs and facilities and are two avenues that can be used for obesity

prevention.

Key words: Down Syndrome, Feeding Difficulties, Obesity.

Mohamed BA, Alhamdan AA, Samarkandy MM. Dietary Practice and Physical Activity in

Children with Down Syndrome and their Siblings in Saudi Arabia. Canad J Clin Nutr 2013;

1(1): 35-46.

INTRODUCTION

Down‘s syndrome (DS) is characterized by

mental and growth retardation associated

with genetic anomalies. DS is affecting

approximately 1 in 800 live births without

predilection for race or socioeconomic

class (1). In Kingdom of Saudi Arabia

(KSA), the incidence of DS births is

around 1.8 for every 1000 babies born

alive (2). In Arab countries it ranges from

1.93–3.5/1000 live birth (2). Overall, the

incidence worldwide ranges from 1.25–

1.67/1000 live birth (3). Feeding

difficulties and inappropriate nutrition are

common problems among children with

Down‘s syndrome (4, 5). Little is known

about the causes of feeding problems in

infants with DS. These may be associated

with low muscle tone, which also affects

the strength, mobility and range of motion

of the oral muscles and can result in weak

sucking, swallowing, lip closure, and

tongue protrusion, and gastroesophageal

reflux (5, 6). The combination of reduced

oral space and low muscle tone can result

in tongue protrusion (5). True

macroglossia can sometimes occur in

infants with Down syndrome, which

results in additional problems with

breathing, chewing and later on with

speech development. Narrowed nasal

passages and increased respiratory

secretions interfere with nasal breathing

and oral feeding in infants with Down

syndrome. As a result of the early feeding

problems in infants with Down syndrome,

and the emotional responses of parents to

Page 35-46



Pag

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the often unexpected diagnosis of Down

syndrome, early mother-infant attachment

may be disrupted and breast feeding is

often not possible (7).

Feeding disorders in children with Down

syndrome may have important long-term

health consequences, including growth

deficits and decreased performance on

tests of academic and cognitive

performance. Some studies estimate that

up to 80% of children with DS have

difficulties related to food or feeding (7-9).

The attainment of feeding milestones in

DS is also delayed by 10–35% depending

on the age of assessment (10).

Furthermore, a delay in the age of

achievement of motor milestones and the

poor gross motor performance of children

with DS may limit physical activity during

infancy and childhood and may further

decrease physical activity levels during

childhood (4, 11, 12). The feeding practice

and physical activity for children with DS

is not well defined particularly in Saudi

Arabia. To the best of our knowledge this

is the first study to assess the feeding

problems and physical activity in DS

children of Saudi Arabia.

SUJECTS AND METHODS

Al-Nahda Schools for DS and the DS

Charitable Association are the only two

schools for DS children in Riyadh city,

Saudi Arabia. The DS children in these

schools are distributed in class rooms

according to their chronological ages. The

approval to conduct the study was

obtained from the school board of these

two schools. One hundred and eight

families were enrolled in the study.

Consent of all families was obtained, and

the study objective was fully explained to

them. The study group included pre

pubertal DS boys and girls (n=108)

clinically and/or cytogenetically proven to

be ailing with DS. Healthy siblings, closest

in age to the DS children were used as a

control group (n =113). Some of the

siblings were twins. The reason for using

siblings as controls in the study was to

ensure quite similar environmental

backgrounds. All the DS children included

in our samples were living with their

parents and had at least one sibling; all

siblings of DS children were living in the

same house. The study was conducted

during the period of February-May 2011.

Thirteen illiterate mothers and 10 mothers

with elementary education that were not

able to fill the questionnaire and were

personally interviewed by the observer in

the school class rooms. The history of

infant feeding among DS children and the

siblings was obtained including type of

feeding and duration of breast feeding.

Nutrition feeding problems were recorded

for the DS children and the siblings, such

as difficulties in using utensils, chewing

and swallowing difficulties, food rejection

and refusal. Physical activity status for the

DS children and matched siblings were

collected from their parents. Food

frequency questionnaire was used to

evaluate dietary habits. The parents were

interviewed for daily details of food

consumed by their children.

Data entry and analysis was carried out

using SPSS 18. Results were presented in

number (n) and in percentage (%), and

were presented as mean values ± standard

deviation (SD). For testing statistical

difference between the DS children and

siblings, and Chi-Square test were used. P-

value less than 0.05 (P<0.05) was

considered statistically significant.

RESULTS

Table 1 illustrates that the mean age is

comparable between DS children and their

siblings. DS children were significantly

shorter and had a higher BMI than their

siblings. The DS children weight less than

their siblings, but the difference did not

reach the significant level. Table 2 shows

history of infant feeding of DS children

and their siblings. The main interesting

results were that during infancy period,

36.4% of DS children were bottle fed



Pag

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compared to only 5.5% of siblings. Almost

half of the breast-fed DS children were fed

for duration of less than 6 months

compared to 36.5% of the breast-fed

siblings. The percentage of DS cases that

were not encouraged by the hospital to be

breast fed (35.4%) was greater than the

siblings (15.8%). The delay in introducing

solid foods was markedly higher in the DS

children compared to the siblings.

The percentage of DS children

experiencing difficulties in using utensils

and chewing/swallowing was almost ten

times higher than the control siblings

(Table 3). The percentage of DS children

who experienced food rejection was

almost twice the percentage of those

siblings and the percentage of DS children

who experienced vomiting after meals was

almost three times higher than the siblings.

About one third of DS children did not eat

with their families as compared to only

15% of siblings.

Table 4 represents, the percentage of

mothers preparing meals for DS children

was lower compared to the siblings, but

the difference did not reach the significant

level. The frequency of eating fast food

was lower in the DS compared to the

siblings. The barrier for conducting

physical activity among DS children were

(dislike of parents to physical activity,

weather condition, lack of facilities or

space, lack of time, and embarrassment),

and it was reported that 73% of the DS

children did not exercise as compared to

44.2% of siblings, and in the group of

children who exercised, only 13.8% of the

DS children spent more than 30 minutes in

exercising as compared to 79.4% of

siblings.

Table 5 shows the frequency of

consumption of some food items for the

DS children and siblings. The main

interesting result was that the weekly

consumption of meat was lower in the DS

cases as compared with the siblings. Also

fruits and vegetables consumption was

lower in the DS children compared with

the siblings. In respect to the consumption

of dairy products, the consumption of

whole milk was lower in the DS children

compared to the siblings, while the

consumption of low fat or skimmed milk

was higher in the DS children as compared

with the siblings. No significant difference

was detected between DS children and

their siblings for 1-3 times consumption

for all items (χ2= 7.66, P = 0.57), for 4-6

times consumption (χ2= 31.83, P = 0.0002)

and rarely (χ2= 67.09, P = 0.0001), a

significant difference was observed

between DS children and their siblings.

DISCUSSION

Improved medical care has played a role in

improving health quality and longevity of

DS children. In USA, the average age

mortality has increased from 25 years in

1983 to 49 years in 1997, with

approximately 44% surviving to age 60

years and 13% to age 68 years (13, 14).

Longer life expectancy imposes the need

to address dietary intake and feeding

difficulties faced by DS cases. In this

study, a high rate of bottle feeding in

infancy was observed with 36.4% of DS

children bottle fed compared to only 5.5%

of the siblings. This may be attributed to

the lack of breast feeding encouragement

from hospitals which may have led to the

result that DS children being more

frequently bottle fed as compared to the

siblings. Furthermore, poorer motor

function of the mouth and generalized

muscular hypotonia can contribute to less

breastfeeding (6, 15, 16). Decreased

muscle tone is associated with problems

starting sucking, a poor lip seal, slow

sucking and swallowing reflex and

uncoordinated suck/swallow/breathing

pattern. These conditions may contribute

to coughing, choking, aspiration, milk

escaping through the nose which may

result in incomplete feeding (17). Our

results showed that DS children are breast

fed on average in a less duration compared



Pag

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to the siblings. Breast feeding is initiated

in less than 50 % of newborns with DS

(18). DS infants have poor sucking ability

which makes establishment of breast

feeding more difficult, since they have

muscles that are poorly developed, thus

making it difficult to position the infant

during breast feeding, which will require

longer breastfeeding sessions. DS children

may also have problems in their mouth,

such as a too-large tongue or a flat palate,

which can affect the depth of latch on the

breasts (16, 18), this poor sucking will

discourage the mother from breastfeeding

as it takes a great deal of patience to teach

the baby to suck properly (and strongly) to

obtain a milk ejection reflex and to

stimulate the milk (6). In our study, Down

syndrome babies were less frequently

breastfed (64.6%) compared with their

siblings (84.2%) during hospital

admission. Previous studies reported that

the main reasons cited by mothers were

infants‘ illness, frustration and depression.

It was shown that the feeling of

depression, grief, guilt or disbelieve can

make a new mother question whether she

should keep the baby or let alone nurse

him (19).

DS children in this study were introduced

to solid foods at a much later stage

compared to the siblings, which is in

agreement with other findings (8, 9).

Feeding difficulties like food rejection,

chewing and swallowing are common in

children with DS (22-24). In our study,

more than half of DS children were having

difficulties in using utensils, 33% had

chewing problems and almost three fourth

of them had some kind of food refusal.

Some authors attributed this feeding

difficulty to low muscle tone, reduced oral

space, presence of heart defects, slow

sucking reflex, slow swallowing reflex,

low birth weight, choking, and narrowed

nasal passage and increased respiratory

secretion (2,4,5,9). It was reported that

feeding problems in infants with Down

syndrome change as they grow older (10).

The infants may experience problems with

transition from breast/bottle feeding to cup

feeding, and from liquids to solids which

can result in inadequate lip closure, poor

chewing ability and choking. Regarding

food frequency consumption, the main

interesting results were that the daily

consumption of meat like lamb, chicken

and fish was lower in the DS children as

compared to the siblings, and fruits, juices

and vegetable daily consumption was

lower in DS children compared to the

siblings. This is in agreement with

Hopman et al., who reported that children

with Down syndrome consume a

substantial amount of products that

requires less mastication (22). It was

suggested that fruits and vegetables are

rejected or not offered to DS children (10).

The present study showed that some

parents were isolating their DS children

from other family members, as it was

found that the percentage of DS children

not sharing meals with the family was

significantly higher as compared with their

siblings. Furthermore, the percentage of

the mothers preparing meals for their DS

children was lower compared to siblings.

Several studies reported that DS mothers

feel anxious, frightened, guilty, angry and

in rare cases, suicidal (23 -26). Other

major observation seen in DS children is

less time exercising. International research

has shown that one of the major influences

on children with DS is parental

involvement and support in physical

activity (27, 28). Previous studies on

physical activity in Saudi Arabia showed

that inactivity prevalence was 97.3% in

Riyadh, with about 70% of preschool

children, about 60% of elementary and

96.1% of married being physically inactive

(29,30). Poor weather has been identified

as an environmental barrier to being

physically active. In Riyadh the weather is

a typical desert climate, being very hot in

summer and very cold in winter, beside

apartments are small and inadequate areas

are available to play at.



Pag

e39

Work leaves no time for physical activity.

Parents come late from work, work double

shifts or have social and family obligations

beside in Saudi culture; women are not

allowed to go out unless she is

accompanied by the husband or a very

close relative. A very strong barrier is the

presence of grief, even shame or

embarrassment for having a DS child (25,

28).

CONCLUSION

The presence of a sibling as a control

group to the DS children decreases the

potential for confounding by

environmental and genetic factors, and

also decreases the recruitment bias of

using healthy controls. The results

suggest that parents can play a key role

in the participation of children with DS

in physical activity as this may be one

avenue for obesity prevention. Children

with DS need specially designed

program and facilities. Another avenue

is controlling feeding practice. Mothers

of DS children should be trained and

given advice to make decision regarding

feeding options. Attention should be

given to the age at which solid food is

introduced as late introduction can be

harmful to oral-motor development and

may be responsible for developing

malnutrition.




study.






manuscript.

Study Limitations

Sample size number and the shortage in

records regarding medical condition status

for DS children were the major limitations.

Acknowledgements

The authors deeply extended their

appreciation to the parents of the enrolled

children for dedicated effort to complete

this study.

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Table 1: Age and anthropometric assessment of study subjects

Parameter

DS children

Mean ± SD

Siblings

Mean ± SD

P- value

Age (years)

Boys 8.2 ± 1.7 8.9 ± 1.4 0.115

Girls 7.9 ± 1.5 8.1 ± 1.6 0.523

Weight (kg)

Boys 22.2 ± 7.7 26.1 ± 7.8 0.054

Girls 20.4 ± 6.3 21.5 ± 4.9 0.921

Height (cm)

Boys 108 ± 9.9 124 ±12.9 0.001

Girls 105.75 ± 8.3 115.1 ± 10.1 0.001

BMI (kg/m2)

Boys

Girls

19.1 ± 4.1

18.5 ± 3.1

16.9 ± 3.4

16.3 ± 1.9

0.013

0.039



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Table 2: Prenatal nutrition of study subjects

Prenatal Nutrition DS Children Siblings

P - Value n % n %

History of infant feeding

Only breast feeding

Breast fed + Formula milk

Bottle feeding

23 21.5 42 38.2

0.0001 45 42.1 62 56.4

39 36.4 6 5.5

Duration of breast feeding

Less than 6 months

6 – 12 months

More than 12 months

33 48.5 38 36.5

0.02 22 32.4 33 31.7

13 19.1 33 31.7

Hospital encouragement of breast feeding

No

Yes

35 35.4 16 15.8 0.002

64 64.6 85 84.2

Age of introduction of solid food

4 months

5 months

6 months

7 months or more

11 11.5 56 53.3

0.0001 25 26 34 32.4

19 19.8 12 11.4

41 42.7 3 2.9



Pag

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Table 3: Difficulties associated with eating meals and food refusal of study subjects

Eating Meals Practice

DS Children Siblings

P –Value

n % n %

Difficulties in using Utensils

No 62 57.4 108 95.6 0.0001

Yes 46 42.6 5 4.4

Vomiting after meals

No 81 75 104 92 0.0006

Yes 27 25 9 8

Chewing/swallowing difficulties

No 72 67 110 97.3 0.0001

Yes 36 33 3 2.7

Food rejection

No 30 28 73 65

0.0001

Yes 74 72 40 35



Pag

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Table 4: Trends of eating and physical activity of the study subjects

Trends of Eating and Physical

Activity DS Children Siblings

P-Value

n % n %

Eating meals with the family

0.0005 No 38 35 17 15

Yes 70 64.8 96 85

Meal preparation

0.275 Mothers 65 60 76 67

Others 43 40 37 33

Times/week of eating fast food

1 64 59.3 51 45.1 0.006

≥ 2 44 40.7 62 54.9

Physical exercise

No 79 73.1 50 44.2 0.001

Yes 29 26.9 63 55.8

Duration (minutes)

< 30 25 86.2 13 20.6

0.001 ≥ 30 4 13.8 50 79.4



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Table 5: Weekly consumption of selected food items of study subjects

Food Items

Frequency of consumption

DS Siblings

1 - 3 4 - 6 Rarely 1 - 3 4 - 6 Rarely

Meat 55 (51) 2(1.8) 51 (47.2) 70 (61.9) 33(29.2) 10(0.09)

Chicken 93 (86.1) 13 (12) 13 (12) 2 (1.9) 81 (71.7) 15 (13.3) 1 (0.9)

Fish 93 (86.1) 1 (0.9 14 (13) 94 (83.2) 1 (0.9) 8 (7.1)

Egg 92 (85.2) 11 (10.2) 5 (4.6) 95 (84.1) 15 (13.3) 3 (2.7)

Whole milk 62 (57.4) 15 (13.9) 31 (28.7) 75 (66.4) 31 (27.4) 7 (6.2)

Low fat milk/skimmed 47 (43.6) 44 (40.7) 17 (15.7) 31 (27.4) 32 (28.3) 50 (44.2)

Cheese 86 (79.6) 19 (17.6) 3 (2.8) 94 (83.2) 18 (15.9) 1 (0.9)

Rice/macaroni 96 (88.9) 11 (10.2) 1 (0.9) 95 (84.1) 17 (15) 1 (0.9)

Fresh vegetables 68 (62.9) 18 (16.7) 22 (20.4) 73 (64.6) 31 (27.4) 9 (8)

Fresh fruits 75 (69.5) 16(14.8) 17 (15.7) 81 (71.7) 32 (28.3) 0



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Generation of Insulin Producing Cells using Mesenchymal Stem Cells

Derived from Bone Marrow of New-Zealand White Rabbits

Abdelaziz A. Mohamed

1*, Manal M. Saad

1, Sherif H. Abdeen

2, Mona K. Marei

1

1Tissue Engineering Laboratories, Faculty of Dentistry, Alexandria University, Egypt.

2Department of Zoology, Faculty of Science, Mansoura University, Egypt.

*Corresponding Author: Dr. Abdelaziz Mohamed. Email: [email protected]

ABSTRACT

Background: The utilization of stem cell trans-differentiation into insulin-producing

cells (IPCs) would provide potential promising therapy for diabetes mellitus (DM).

Objective: The study was aimed to investigate the differentiation potential of rabbit‘s

bone marrow-derived cells into insulin producing cells. Methods: Bone marrow-derived

mesenchymal stem cells (MSCs) were obtained from three New-Zealand male white

rabbits and propagated in a primary culture for 14 days in low glucose Dulbecco's

Modified Eagle's Medium (DMEM), harvested and subjected to another three passages

encompassing 21 days. After that, MSCs were functionally defined by their ability to

differentiate into osteoblasts. This was achieved by incubation with the DMEM

containing 10-7

M dexamethasone, 10 mM β-glycerophosphate and 50 µl/ml of ascorbic

acid. Osteogenic differentiation was followed up at days 2, 4, 7 and 9 by staining cells

with alizarin red S and vonKossa. Results: It was found that the onset of differentiation

was at day 7 and continued at day 9. On the other hand, another patch of MSCs were

induced into insulin-producing cells by two step incubation. The first with high glucose

serum-free DMEM containing 0.5 mmol/L β-mercaptoethanol for three days and the

second follows by incubation with the same medium containing 10 mmol/L

nicotinamideinstead of β-mercaptoethanolfor 18 days. Trans-differentiation was followed

up at days 4, 9, 14 and 21. It was found that the cells have trans-differentiated into

insulin-producing cells starting from day 14 and continued in the subsequent days as

judged by their affinity to stain with diphenylthiocarbazone (dithizone or DTZ) at days

14 and 21. Conclusion: The results would provide some insights of using rabbit's bone

marrow as a source of MSCs with their differentiation potentials. This might help for the

development of a future stem cell therapy for diabetes as well as several other human

diseases.

Key words: Mesenchymal Stem Cells, Insulin-producing Cells, Diabetes Mellitus

Mohamed AA, Saad MM, Abdeen SH, Marei MK. Generation of Insulin Producing Cells

using Mesenchymal Stem Cells Derived from Bone Marrow of New-Zealand White Rabbits.

Canad J Clin Nutr 2013; 1(1): 47-66.

Page 47-66




Pag

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INTRODUCTION

Stem cells are undifferentiated cells

which have the ability to renew

themselves through mitotic cell division

and differentiate into a diverse range of

specialized cell types (1). Of these cells,

mesenchymal stem cells (MSCs) have a

large capacity for self-renewal while

maintaining their multipotency. MSCs

display multiline age differentiation

potentials into a variety of cells such as

osteoblasts, adipocytes and chondrocytes

as well as myocytes and possibly

neuron-like cells (2). They are capable

of suppressing immune responses and

offer therapeutic potential for achieving

transplantation tolerance (3).

Accordingly, MSCs have been

considered as an appropriate source for

cell and gene therapy tools for treatment

in a number of injuries, congenital and

degenerative diseases including; spinal

cord injury (4), osteogenesis imperfecta

(5), stroke (6), parkinsonism (7) and

diabetes mellitus (8).

Diabetes mellitus refers to a group of

chronic metabolic diseases characterized

by hyperglycemia due to defects in

insulin secretion, insulin action, or both

(9).Type I, insulin-dependent, diabetes is

the result of autoimmune destruction of

insulin-producing pancreatic β-cells. On

the other hand, in type two or non-

insulin-dependent diabetes, insulin

production is inadequate mainly because

of the peripheral insulin resistance and

subsequent β-cell apoptosis. Diabetes

and its devastating effects, which include

retinopathy, nephropathy, stroke and

heart attack, afflict more than 194

million people worldwide. According to

the World Health Organization, these

numbers will be more than double by

2030 (10, 11).

Survival of patients with insulin-

dependent diabetes relies on recurring

insulin delivery, which does not cure the

disease or prevent diabetes-associated

maladies. Although, Type 2 diabetes can

be managed through a combination of

diet, exercise and prescription of drugs,

almost 30% of the affected people will

require frequent administration of

insulin. Therefore, the development of

therapies to replace insulin regimens is

highly desirable. To that end, islet

transplantation has afforded promising

results, with some patients experiencing

insulin independence for more than 5

years after the initial procedure (12).

Ongoing procedural improvements are

underway to increase the time span of

liberation from insulin and reducing the

side effects due to immunosuppressant.

However, the scarcity of available donor

tissues hinders wide application of

pancreas/islet transplantation. Thus, the

hope stands in finding renewable sources

of islet β-cells such as pancreatic β-cell

lines, embryonic stem cells (ESCs),

adult progenitor cells (APCs),

regenerating native islet cells (13) and

MSCs (14).

From all of the previously mentioned

literatures, it was therefore of interest to

carry out experiments investigating the

differentiation potential of rabbit's bone

marrow-derived stem cells into insulin-

producing cells as a first step for their

future use in islet transplantation

purposes.



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MATERIALS AND METHODS

Abbreviations

The following abbreviations are used;

Adult progenitor cells (APCs), deionized

distilled water (ddH2O), diabetes

mellitus (DM), dimethyl sulfoxide

(DMSO), diphenylthiocarbazone

(dithizone or DTZ), Dulbecco's

Modified Eagle's Medium (DMEM),

embryonic stem cells (ESCs), fetal

bovine serum (FBS), hours (hr), insulin I

and II, glucose transporter-2 (Glut-2),

insulin-producing cells (IPCs), intra-

muscular (IM), mesenchymal stem cells

(MSCs), minutes (min), mononuclear

cells (MNCs), pancreatic duodenal

homeobox-1 (Pdx-1) and phosphate

buffer saline (PBS).

Animal work

All animal experiments were performed

according to the guidelines of the

National Institutes of Health (NIH) for

laboratory animal use (15). All

procedures applied in this study were of

no pain category (category C).

Aspiration bone marrow-derived cells

Bone marrow aspiration was performed

according to the method of Horan et al.

(16).Three male New-Zealand white

rabbits (obtained from the research farm,

Faculty of Agriculture, Alexandria

University) were anesthetized by intra-

muscular (IM) injection with 5 mg/kg

xylazine as a preanaesthetic medication.

After 5 min, animals were intra-

muscularly injected with 30 mg/kg

ketamine-HCl. All surgical procedures

were performed in a biological safety

cabinet (Nuaire biological safety class II,

T-Telestar, Spain) with surgery room

disinfected overnight by UV light. The

fur of the thigh was shaved and skin

disinfected. Skin incision was made to

expose the rabbit's femur. A hole was

drilled in the femur using a surgical

rose-head bur. A sterile plastic aspiration

needle containing 8 µl heparin/ml

sample was used. The marrow sample

was kept in a sterile package till

transferred to the cell culture laboratory.

The surgical wound was sutured and the

rabbit was taken back to the animal care

unit. All animals tolerated the surgical

procedures for bone marrow aspiration.

No significant changes in body weight,

no postoperative infections were

observed.

Cultivation media

All buffers, media and their supplements

were purchased from BioWhittkers®

Lonza, Verviers, Belgium unless

otherwise noted. These media included:

Basal medium

[Low glucose Dulbecco's Modified

Eagle's Medium (DMEM) with 4.5

mmol glucose/L and supplemented

with10% (v/v) fetal bovine serum

(FBS),1% L-glutamine,10 IU/ml

penicillin/streptomycin and 20 mM

HEPES]. Ostogenic differentiation

medium [Low glucose DMEM with4.5

mmol glucose/L and supplemented with

10% FBS, 1% L-glutamine, 1%

penicillin/streptomycin, 20 mM

HEPES,10 mM β-

glycerophosphate(Sigma, USA),50

µg/ml ascorbic acid (Sigma, USA)

and10-7

M dexamethasone (Sigma,

USA)].

First step trans-differentiation medium:

Serum-free high glucose DMEM with 25

mmol glucose/L and supplemented with

20 mM HEPES,1% L-glutamine,1%

penicillin/streptomycin and 0.5 mmol/L

β-mercaptoethanol (Sigma Aldrich Co.,



Pag

e50

Germany). Second step trans-

differentiation medium: Serum-freehigh

glucose DMEM with 25 mmol glucose/L

and supplemented with 20 mM HEPES

buffer,1% L-glutamine,1%

penicillin/streptomycin and10 mmol/L

nicotinamide (Sigma Aldrich Co.,

Germany).

Media preparation

The powder supplements of the media

were dissolved in deionized distilled

water (ddH2O) obtained from Barnstead

water deionization system (Barnstead-

Thermolyne, USA). In case of using

another dissolvent, it was sterilized by

membrane filtration through 0.22 μm

microbiological filter system (TPP-

Europe, Switzerland). After adding the

supplements, the pH of the medium was

adjusted at 7 and medium was then

sterilized by membrane filtration. A

sterility test was performed for 3 days at

37C. Medium contamination was

detected depending on the color change

of the phenol red, which gives a yellow-

orange color through the change in pH to

acidic value as a function of the

microbial growth. A sample was also

examined microscopically after passing

the sterility test. All media were stored at

4C until being used.

Isolation of rabbit's bone marrow-

derived mesenchymal stem cells

The method of Sun et al., (17) was used

with minor modifications. Bone marrow

samples of about 1.5 to 2 ml were

aspirated from each rabbit and

transferred to the cell culture laboratory

in a sterile package. Inside a biological

safety cabinet class II (Thermo electron

corporation, Germany) samples were

diluted with a double volume of the

basal medium, collected by

centrifugation at 390 g for 7 min at room

temperature and washed twice with

phosphate buffer saline, pH 7.4 (0.0067

M PBS without calcium and

magnesium).

The samples which have clots or bone

marrow tissue were filtered through 200

µm filter (Sigma Aldrich Co., Germany)

using PBS for washing and liberation of

the trapped cells in the tissue. Bone

marrow mononuclear cells (MNCs) were

then collected by centrifugation, washed

twice with the basal medium and

resuspended in one ml of the same basal

medium, counted (10 l of cells + 10 µl

of 0.4% methyl violet + 180 µl glacial

acetic acid) and tested for viability using

hemocytometer. MNCs were then

seeded in polystyrene coated T-flasks at

a density of 5.5 x 106cells/cm

2and

incubated at 37oC, 5% Co2 and 95%

humidity. Except for the first four days

which were required for cell adhesion,

the cells were fed twice a week by

washing with pre-wormed PBS then

adding fresh medium. The cells were

allowed to proliferate till reaching the

100% confluence in 14 days (the

primary culture).

Only the cells which attach to the

polystyrene surface were allowed to

grow while suspended ones were

discarded during washing. Cell

morphology was followed up daily using

a phase contrast inverted microscope

(NIKON-Japan) using (SIS) software

system for capturing and analysis of

images. All cultures were independently

repeated for three times.

Confluent monolayer of cells were

harvested by trypsinization using

trypsin/EDTA buffered solution 6 ml of

0.5% trypsin, 200 mg/L versene EDTA



Pag

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(Gibco/Invitrogen, Germany) at 37oC for

10 min. The suspended cells were

rapidly neutralized with a double volume

of the basal medium to stop the action of

trypsin. The cells were then collected by

centrifugation, washed twice with pre-

wormed PBS, counted and tested for

viability. The cells were then reseeded in

fresh medium at a density of 1400 cells

per cm2 in a new cell culture T-flasks.

The cells were allowed to grow for three

subsequent passages of 7 days each.

After passage three, MSCs were

subjected to osteogenic differentiation

and trans-differentiation into insulin-

producing cells (18).

Estimation of viable cell concentration

Viable cell concentration was

determined using trypan blue exclusion

method (19). Ten l of cells were diluted

to 1:2 in 0.4% trypan blue stain. The

hydrophilic trypan blue diffuses through

cell membrane of the dead cells. Hence,

the viable cells appear bright while the

unviable ones appear blue. Cell viability

was determined according to the

following equation: Cell viability =

Total number of viable cells/Total cell

count ×100

Cryopreservation of bone marrow-

derived cells

At the end of each culture, a separate

patch of cells were trypsinized and

collected for cryopreservation (20). The

cells were cryopreserved at a density of

1×106 cells/ml in freezing medium; basal

medium with 25% FBS and 5% v/v

dimethyl sulfoxide (DMSO). The cells

were then stored at -156ºC in cryo-vials

(Nunc, Wiesbaden, Germany) in the

vapor phase of a liquid nitrogen locator

(Cryo Biological Storage System

Locator 4 plus thermolyne, USA).After

several pilot trials, cryopreservation of

cells was optimized in 5% DMSO as a

cryoprotectant. This protocol yielded the

highest cell viability of 69% upon

revitalization. In order to revitalize the

cells, cryogenic-vials were warmed in a

water bath at 37oC for 2 min and

resuspended rapidly in FBS, centrifuged

for 5 min, washed twice with PBS,

counted, tested for viability and

resuspended in the basal medium.

Counting and testing viability were done

before and after cryopreservation as a

routine procedure.

Characterization of rabbit's bone

marrow-derived MSCs

Rabbit's bone marrow-derived MSCs

were characterized by their induction

into osteogenic differentiation using the

method of Nadriet al.,(21). The cells

were incubated with the osteogenic

differentiation medium at the end of

passage three, especially, when they

have reached 80-90% confluency. The

cultures, in 6-well plates, last for two

weeks with cells fed twice a week. Cell

morphology was followed up daily. To

detect the onset of osteogenic

differentiation, cells were stained with

alizarin red S and von Kossa at days 2,

4, 7 and 9.

Alizarin red S staining of the

differentiated osteoblasts

Calcium depositions of osteoblasts were

detected through its reaction with

alizarin sulphate with scarlet to red color

indicating positive results (22). For

staining, the cells were washed with pre-

warmed PBS, fixed in 4% formaldehyde

for 30 min. The fixative was then

removed and the cells were washed

using distilled water. The alizarin red S

stain pH 4, (2% alizarinsulphonate;



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LobachemieCo., India) was added to

cover all the cells for approximately 5

min at room temperature. The stain was

then discarded and the cells were washed

again with distilled water to remove the

excess of stain then air dried. Red

patches in the tested cultures were

examined microscopically. Cells

propagated in the basal medium were

included as control cultures.

vonKossa staining of the differentiated

osteoblasts

vonKossa staining was applied to

confirm the osteogenic differentiation of

MSCs (23). Like the staining protocol of

alizarin red S, the cells were washed

with pre-warmed PBS, fixed for with 4%

formaldehyde 30 min and washed with

distilled water. The cells were then

incubated with 1% aqueous silver nitrate

solution (Fisher scientific-UK) in a clear

glass coplin jar placed under a 60-100

watt light bulb for 1 hr. After that, the

cells were carefully washed with

distilled water to eliminate all the debris

of silver nitrate and incubated with 5%

sodium thiosulfate for 5 min. The

solution was discarded and the cells

were washed again with distilled water

and air dried. The progress of the

reaction was followed up visually. The

staining intensity of the brown to black

color was a reference for the presence of

calcium produced by the differentiated

osteoblasts.

Trans-differentiation of MSCs into

insulin-producing cells

At the end of passage three, especially

when MSCs have reached 80-90%

confluence, the cells were induced into

insulin producing cells through two

steps. The first step included the

incubation of the cells with the 1ststep

trans-differentiation medium for three

days. This was followed by a careful

wash with pre-warmed PBS and

incubation with the 2nd

step trans-

differentiation medium for 18 days. All

over the two steps, the cells were fed

twice a week (17).

Staining of insulin-producing cells

with dithizone

Dithizone (DTZ), a zinc-chelating agent,

is known to selectively stain pancreatic β

cells by crimson red color. In the

secretory granule of a mature insulin-

producing cell, every 6 insulin molecules

are coordinated by a Zn atom giving the

red color. For staining, the cells were

incubated with DTZ stain (Lobachemie

Co., India; 10 mg/ml DTZ in DMSO

diluted to 1:100 in the basal medium) for

30 min. The cells were then carefully

washed with pre-wormed PBS for three

times. The staining result was monitored

under an inverted microscope. After 5

hours, the stain was completely

disappeared and the cells were able to

grow successfully (24).

Data analysis

Statistical analyses were performed with

one-way ANOVA followed by SPSS

software version 10 (SPSS Science,

Chicago, IL, USA) analysis. Data (mean

± SD) were considered statistically

significant at a value of P < 0.05.

ANOVAtest was applied to compare the

growth kinetics during the different time

points of the expansion period i.e. the

primary culture and the first two

passages.



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RESULTS

Isolation of bone marrow-derived

mesenchymal stem cells

Isolation of bone marrow-derived MSCs

required a primary culture of 14 days

and three subsequent passages of 21

days. This can be summarized as

follows; bone marrow samples of 1.5 - 2

ml were processed for isolation of

mononuclear cells (MNCs) giving rise to

the mean numbers of cells 3.9×107.

MNCs were then seeded in a primary

culture at a density of 5.5×105 cells/cm

2

giving a yield of 15.6×103 cells/cm

2 at

the end the primary culture (day 14).

Whereas, the number of cells seeded in

passage one and two was 1.4×103

cells/cm2 and the number of cells yielded

was 8.4×103 and 7.2×10

3cells/cm

2 for

passages one and two, respectively. The

cells were allowed to grow in passage

three similar to that of passages one and

two with one difference of being

differentiated or trans-differentiated at

the 100% confluency of passage three

without transfer to a next passage (Table

1). Except for day zero of the primary

culture, comparison of the growth

kinetics of all culture during the

expansion period (the first 5 weeks)

revealed that all cultures were

significantly grow (P < 0.05) starting

from day 2 after reseeding.

In the primary culture, MSCs were

allowed to adhere to the polystyrene

tissue culture flask surface in the first

four days without media change.

Whereas, at day five all of the co-

cultured red blood corpuscles (RBCs)

and cell debris were discarded with the

washing step prior to the media change.

At day five, MSCs showed an

anchorage-dependent growth with their

attachment confirmed via the emergence

of expanded cytoskeleton processes. In

the following days, cells proliferate with

increased cell to cell contact giving rise

to cell colonies. By the end of the

primary culture, at day 14, the cell

proliferation reached about 90%

confluence. Thus, the cells were

trypsinized and reseeded in another

culture (Figure 1).

In passage one and unlike the primary

culture, the cells adhere in few hours and

overnight incubation was sufficient to

make the cells to completely adhere in

day 1. However in day 2, the cells

started to contact with each other. In the

subsequent days, the cells proliferated,

forming colonies and their morphology

changed to the spindle-shape. The cells

reached the 100% confluence in only 7

days (Figure 2). After that, the cells were

collected by trypsinization and reseeded

in a new cell culture flask for another

passage. The number of cells at the end

of passage one was 8.2×103 cells/cm

2

with doubling time of 21.6 hr. The

viability after harvesting was 98%.

However in passage two, the cells

showed similar manner of cell growth.

They showed substratum adherence,

migration, cell-cell contact, colony

formation and confluence. The cell yield

of passage two was 7.2×103 cells/cm

2

and doubling time was 17.63 hr.

Characterization of MSCs by

osteogenic differentiation

Cells were incubated in conditioned

medium of dexamethasone, β-

glycerophosphate and ascorbic acid.

They were following up daily with an

inverted microscope. The cells were

stained with alizarin red S and von

Kossa stains in days 2, 4, 7 and 9 to

determine the onset of osteogenic



Pag

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differentiation. All tested cultures

showed positive results starting from day

7. However, controls showed negative

staining all over the four time points.

On the other hand, the morphology of

the cells under the stress of osteogenic

differentiation showed the spindle-shape

in the tested cultures. The controls

showed some aspect of over confluent

appearance. The difference in cell

morphology between the tested and

control cultures during the first few days

till day 7 was not clear but with staining

the discrimination was possible (Figure

3).

Trans-differentiation of MSCs into

insulin-producing cells

MSCs were induced into insulin-

producing cells via two steps. The first

step included the incubation of cells with

high glucose serum-free DMEM

containing β-mercaptoethanol. Upon

trans-differentiation, the cells showed

aggregated colonies. The first step of

differentiation was extended to 3 days

then the second step follows at day 4 by

replacing β-mercaptoethanol with

nicotinamide. At day 4, the colonies

were formed and developed in the

subsequent days. At day 14, the

morphology of cells was changed to the

rounded to oval shape and continued

with this morphology in days 19 and 21

(Figure 4).

The dithizone (DTZ) staining was

applied to test the production of insulin

at different time points including days 4,

9, 14 and 21. While, the tested cultures

showed an affinity to the DTZ stain

(crimson red color) starting from day 14

and continued in day 21, the controls

showed negative results all over the time

of the experiment (Figure 5).

DISCUSSION Mesenchymal stem cells are

multipotentstem cells that can

differentiate into a variety of cell types

(25). Bone marrow-derived MSCs, also

named colony-forming fibroblastic cells

(26), marrow stromal stem cells (27) and

mesenchymal progenitor cells (17), have

been isolated and characterized from

many species including: rats, cats, dogs,

baboons and rhesus monkeys, rabbits,

pigs, goats and sheeps (21). MSCs have

many advantages including: the ease of

isolation, the extensive multi-lineage

differentiation potential (18, 28), the

hypo-immunogenic property which

modulate the lymphocytic functions, the

possibility of allogenic transplantation,

the capability of systemic transplantation

for generalized diseases as well as the

local implantation for the local tissue

defects. MSCs can also be used as a

vehicle for genes in gene therapy

protocols or to generate transplantable

tissues and organs in tissue engineering

protocols. Accordingly, MSCs are

among the first stem cell types to be

introduced in the clinic and several

encouraging clinical trials are under way

to study the efficacy and long-term

safety of therapeutics based on MSCs

(29, 30).

Several recent studies have demonstrated

the feasibility of generating insulin-

producing cells obtained from progenitor

cells of various cellular sources,

including the pancreas, liver and

intestinal epithelium, as well as, the

pluripotent embryonic stem cells of

mouse and human origin. However, even

with the conceptual advances offered by

these findings, some obstacles, such as

the immune rejection and autoimmunity

against newly formed cells derived from

http://en.wikipedia.org/wiki/Multipotent

http://en.wikipedia.org/wiki/Multipotent

http://en.wikipedia.org/wiki/Cellular_differentiation



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pancreatic stem cells, still remain.

Despite their promising potential, it may

also prove difficult to obtain enough

autologous adult stem cells from these

organs. To overcome these limitations,

the possibility of using rabbit bone

marrow derived cells was explored as a

source of insulin-producing cells under

specific in vitro culture conditions. Bone

marrow has been known for years to

represent a safe and abundant source for

large quantities of adult stem cells (13).

In the present study, the source of MSCs

was the bone marrow of New-Zealand

white rabbits. This animal model was

chosen due its availability, the ease of

handling and the ease of choice between

in and out-bred. In case of aspiration of

bone marrow samples from mice or rats,

the animal is often killed before

aspiration. Contrarily, there is no need to

kill the rabbit to aspirate a bone marrow

sample with the advantage of repeating

aspiration within 8 weeks (16).

Several techniques have been developed

to obtain pure cultures of MSCs by the

reduction or elimination of non-MSCs

from bone marrow cultures (31). This

was achieved, in the present study, by

the application of a prolonged expansion

step through which all of the bone

marrow-derived cells pass in a primary

culture for 14 days and three subsequent

passages of 21 days. During this time,

only MSCs, due to their adherent

property, were retained in the culture

while all of the other non-adherent cells

were eliminated by washing during

media change. Moreover, the selective

medium and the polystyrene-coated

tissue culture flasks enhanced the

adhesion properties of the MSCs (21).

Cultures of MSCs is usually

contaminated by different types of non-

MSCs adherent cells like monocytes and

macrophage (32) and as MSCs are more

responsive to trypsin, they were purified

in the present study with an optimized

concentration of trypsin/EDTA (17).

Unlike the primary culture when the

cells needed 14 day to reach the 100%

confluency, the behavior of the cells

changed in the subsequent passages as

they reached the 100% confluency in

only 7 days. This could be owing to the

gradual selection of a learned

mesenchymal cell clones with

proceeding passages (33).

One of the hallmarks of MSCs is their

multi-potency, defined as the ability to

differentiate into several mesenchymal

lineages including; bone, cartilage,

tendon, muscle, marrow stroma and

adipose tissue. Usually, the tri-lineage

differentiation into bone, adipose tissue

and cartilage is taken as a criterion for

the multi-potentiality of these cells (34).

In the current study, MSCs were

differentiated into osteoblasts using a

conditioned medium containing

dexametasone, ascorbic acid and β-

glycerophosphate. The differentiation

was followed up at different time points

including days 2, 4, 7 and 9 through

staining with alizarin red S and von

Kossa stains. The onset of osteogenic

differentiation was at day 7 as judged by

the positive staining affinity of the

calcium depositions of the osteoblasts

with the two stains. These results

confirmed the mesenchymal stem

lineage of the processed cells

(35).

Upon trans-differentiation, the presence

of insulin was evidenced using the

dithizone stain (DTZ) which is a zinc

chelating agent. It does not require prior

fixation and stains insulin molecules



Pag

e56

either in the cells or in the media.DTZ

disappears after 5 hr leaving live cells

able to complete their life cycle (24, 36).

The onset of trans-differentiation was

followed up at different time points

including; days 4, 9, 14 and 21. It was

found that a complete trans-

differentiation was obvious in day 21

while partial trans-differentiation was

observed in day 14. In contrast, there

were no signs of trans-differentiation at

days 4 and 9. Despite DTZ is a vital

stain, the staining intervals were chosen

minimal to decrease the stress applied on

the cells at every staining time.

On the other hand, the microscopic

examination for the trans-differentiation

revealed aggregations like clusters of

insulin producing cells with a diameter

ranging from 100 to 150µm. These

clusters appeared from day 2 of

differentiation and continued to develop

till day 12 where the cells acquired the

spindle-shape. This morphology was

changed to the rounded and oval shapes

at day 14 which may be considered as a

sign for the meso-endodermal

differentiation (37).

There are two key steps in culture

conditions that appear important for

inducing the trans-differentiation of

MSCs cells into insulin-producing islet-

like cells. First, the transfer of cells from

an expansion period in low-glucose

medium to differentiation in high-

glucose medium until certain genes, such

as pancreatic duodenal homeobox-1

(Pdx-1), insulin I and II, glucose

transporter-2 (Glut-2), and islet amyloid

polypeptide, become detectable. Second,

in order for the MSCs cells to become

glucose responsive, further

differentiation and maturation are

required through either in vitro culture

with cell promoting factors, such as

nicotinamide, mercaptoethanol or

transplantation of the cells into diabetic

animals (17, 38). It is well known that

glucose is a growth factor for cells. It

promotes cell replication in vitro and in

vivo and increases insulin content of the

cell (39).

Nicotinamide is a poly (ADP-ribose)

synthetase inhibitor known to

differentiate and increase cell mass in

the cultured human fetal pancreatic cells

(40). It protects the cells from

desensitization induced by the prolonged

exposure to large amounts of glucose. It

have also been demonstrated that

nicotinamide promoted the formation of

fetal porcine islet-like cell clusters and

increased the rates of pro-insulin

biosynthesis in these clusters. Moreover,

the stimulatory effects of nicotinamide

on insulin production and content by

fetal porcine islet-like cell clusters result

from neo-formation of cells through

differentiation (41). Another study

described how nicotinamide-treated

islets derived from the pancreatic

progenitor cell had more insulin and

secreted significantly more insulin than

cultures treated with glucose alone (42).

The present study used the low glucose

medium for cell expansion and for

induction to insulin-producing cells the

medium was changed to high glucose

and this was the first key step in trans-

differentiation. The second key step was

achieved through culturing with the

media containing β-mecaptoethanol then

nicotinamide as cell promoting factors.

In conclusion, the present data suggest

potential uses of MSCs from rabbit's

bone marrow in induction into insulin-

producing cells. This provides hope for a

future development a stem cell therapy



Pag

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for diabetes as well as several other

human diseases and injuries.

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Table 1. Bone marrow-derived cell growth during culture

Total number of living cells

(Mean cells / cm2 ± SD) × 10

3

Primary

culture

Day 0 Day 5 Day 7* Day 9* Day 14*

550 ± 88.8 5.1 ± 0.8 7.77 ± 0.76 11.37 ± 1.01 15.67 ±

0.67

Passage

one


1.4± 0 1.67 ±

0.21 2.93 ± 0.15 5.23 ± 0.31 8.4 ± 0.2

Passage

two


1.4± 0 1.63 ± 0.15 2.3 ± 0.53 4.23 ± 0.31 7.2 ± 0.6

Passage

three

Day 0 Reached the 100% confluency after 7 days.

Characterization by osteogenic differentiation.

Trans-differentiation into insulin-producing cells. 1.4± 0

* Significant cell growth (P < 0.05) compared to the beginning of culture with the

exception of day zero of the primary culture where cultures were compared to the

day 5.



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Figure 1. Photomicrographs of MSCs during the primary culture. Days of follow up

included; (A) day 4, (B) day 5, (C) day 6, (D) day 7, (E) day 9 and (F) day 14. Black

arrow in A refers to a red blood corpuscle and whites arrows in A and B refer to the

adherent MSCs.



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Figure 2. Photomicrographs of MSCs during passage one. Days of follow up

included. (A) Day one and (B) day three, with arrows referring to cell-cell contact.

(C)and (D) are representing the culture at days 4 and 6, respectively. (E) represents the

morphology of the mesenchymal stem cell colony and (F) represents two proliferating

colonies in their way to adhere with arrows representing cell movement.



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Figure 3. Photomicrographs of MSCs at day seven during osteogenic differentiation. (A)and(B) are representing the control and tested cultures, respectively, stained with

alizarin red S. (C) and (D) are representing similar control and tested cultures,

respectively, stained with von Kossa. The orange to red patches in B and the brown to

black patches in D indicate the osteogenic differentiation.



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Figure 4.Photomicrographs of MSCs during the trans-differentiation into insulin-

producing cells. Days of follow up included; (A, B) day 2, (C, D) day 14 and (E, F) day

19. (A, C) are representing the control cultures and (B, D, E, F) are representing the

tested cultures. The control cultures showed an over confluency (arrows). The change in

cell morphology of the tested cultures from the spindle-shape to the oval or rounded-

shape was noted from day 14 and continued to day 19.



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Figure 5. Photomicrographs of dithizone (DTZ) stained MSCs during the trans-

differentiation into insulin-producing cells at day 14 (A, B) and day 21 (C, D). (A, C)

are representing the control cultures and (B, D) are representing the tested cultures. The

control cultures showed an over confluency with negative staining. While, the tested ones

showed the positive staining (crimson red patches).



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The Role of Folate on Colorectal Cancer in the Arabian Gulf

Cooperation Council Countries: An Overview

Mostafa I. Waly,1,4*

, Yahya M. Al-Farsi 2,5

, Allal Ouhtit3

1Department of Food Science and Nutrition, College of Agricultural and Marine Sciences;

2Department of Family Medicine and Public Health,

3Department of Genetics, College of

Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman. 4Department of

Nutrition, High Institute of Public Health, Alexandria University, Egypt. 5Department of

Epidemiology, School of Public Health, Boston University, USA

*Corresponding Author: Dr. Mostafa Waly. Email: [email protected]

ABSTRACT

Driven by the boom of wealth brought by oil discovery and production, the high rate of

urbanization and a steady increase in the per capita income, during the past years, has

improved the socio-economic status of the people in the Arabian Gulf Cooperation

Countries (GCC). This radical economic change has been accompanied by drastic

changes in their lifestyle and food consumption patterns. During the past years, the

regular consumption of traditional foods is being replaced with more Western-style and

ready-made foods. In addition to adopting a sedentary life style in daily life and other

related activities. According to National Health Survies in the GCC countries, high daily

caloric intake, refined carbohydrates and protein were associated with increased risk of non-

communicable diseases including colorectal cancer (CRC). The rate of CRC among GCC

countries is in the rise; however, there is dearth of research exploring it. In particular,

there is lack of research about role of folate on occurrence of CRC despite the recent

flourishing scientific research papers from other parts of the world.

Key words: Colorectal Cancer, Folate, Gulf Cooperation Countries.

Waly MI,Al-Farsi YM, Ouhtit A. The Role of Folate on Colorectal Cancer in the Arabian

Gulf Cooperation Council Countries: An Overview. Canad J Clin Nutr 2013; 1(1): 67-73.

INTRODUCTION

Cancer is considered a leading cause of

death worldwide, accounting for 7.6

million of all deaths from a total of 58

million deaths reported in year 2005 and

the (WHO) estimates that there will be

20 million new cases and 12 million

deaths from cancer alone by the year

2020 (1). Furthermore the WHO

reported that in Western countries, the

rate of colorectal cancer (CRC) has

increased dramatically as compared to

other types of cancers, and this increase

was attributed to westernization in food

choices and dietary pattern as well as

adopting a sedentary lifestyle (1). The

Arabian Gulf Cooperation Council

(GCC) countries include the following

six countries: Oman, United Arab

Page 67-73



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Emirates, Saudi Arabia, Qatar, Bahrain,

and Kuwait (2).

The GCC countries have witnessed rapid

change in many aspects of life during the

last 5 decades because of the boom of

wealth that coincided with the discovery

and production of oil (3). This boom has

transformed these countries from being

traditional nomadic societies to become

affluent metropolitan cities in a very

short time. This rapid change resulted in

an immense effect of the life style of

people, and their social life. This is quite

notable with the proliferation of western

pattern of lifestyle especially in nutrition

and physical inactivity (4). The rapid

socio-economic transition might also

have affected the pattern of cancer in

these countries. It has been quite notable

that incidence rates of cancer have

increased in other countries that went

through rapid industrialization because

of the lifestyle risk factors including

diet, physical activity, obesity which

appears to play an important role in the

etiology of the disease (5).

Recent reports indicate that migrant

populations moved from countries of

low CRC and incidence to the West

adopted the high CRC incidence trend of

the new country (6-8). These

observations suggest that changes in diet

and lifestyle might contribute to the

incidence of CRC, and therefore

research had focused in identifying the

dietary pattern and lifestyle

characteristics that might contribute to

primary prevention of CRC. The

international experts from the recent

World Cancer Research Fund Report,

2008, concluded that CRC is a major

public health problem and modernization

in food choices, sedentary life style and

increased life expectancy are the major

factors that might synergize with

genetics for the epidemic of CRC

worldwide (9).

It is timely to conduct research studies to

elucidate the underpinning risk factors of

cancer, in general, and colorectal cancer,

in particular, in the GCC countries. In

the GCC countries, there are few

published reports about the dietary

pattern and lifestyle characteristics of

CRC among adults. Generally, it has

been noted that the rate of CRC among

GCC countries is considered relatively

low compared to most of western

countries. Nonetheless, the pattern is in

the rise (10). In Oman, CRC ranked third

after breast cancer and stomach cancer

(11). Similar pattern has been observed in

Qatar and Saudi Arabia where it ranked

second after breast cancer, and in both

countries it constituted for 9% of the

newly diagnosed cases (12). In a

comparative review among Arab

countries, it has been noted that the rates

of CRC among Bahrain and Kuwait were

higher than other Arab countries (10).

Sporadic studies about CRC in the GCC

countries do exist. They are mainly

cross-sectional epidemiological studies

that describe the pattern of annual

change of cancer rates in general, and

among them CRC is noted (13). Fewer

studies are case-control studies that

explored underlying risk factors (14). To

our best knowledge, studies that explore

the relation between folate and CRC in

the GCC countries have not been yet

reported. The following sections provide

an overview of the molecular

mechanisms of the role of folate in

occurrence of CRC.

There are several established dietary risk

factors for CRC, largely related to folate

and/or vitamin B12 deficiency (15, 16),

diets from animal sources that are low in

fibre and high in fat (17-19). Meanwhile,



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allium vegetables, fruits, pulses and

foods containing selenium protect

against CRC cancer (20-22). Non dietary

risk factors for CRC include, decreased

physical activity (23), obesity ,a body

mass index (BMI) of greater than or

equal to 30 kg/m2 (24, 25), genetic

susceptibility (26, 27), alcohol (28, 29),

smoking (30), and exposure to

environmental carcinogens that may lead

to proliferation and malignant

transformation of colorectal cells (31,

32).

Folate and vitamin B12 deficiency induce

carcinogenesis in susceptible persons,

via mechanisms that involve cell

signaling, cell division and DNA

methylation. The protective effect of

folate and vitamin B12 is attributed to its

one carbon folate metabolism, including

the methionine cycle that is regulated by

ubiquitous enzyme, methionine synthase

(MS). The mechanism involves the

necessity of 5-methyltetrahydrofolate (5-

CH3-THF) as a methyl donor and

vitamin B12 as a cofactor for MS that

regulates the homocysteine (HCY)

remethylation to methionine and

synthesis of S-adenosylmethionine

(SAM), the universal biochemical

methyl donor for all biological

methylation reactions, including DNA

methylation (33).Numerous studies have

indicated that under conditions of low

dietary intake of folate and vitamin B12,

the methyl supply is low and

consequently the MS activity is low

concomitant with low SAM levels and

DNA hypomethylation, a leading cause

of mutagenesis and abnormal expression

of oncogenes, which have been

implicated in different types of

carcinogenesis (34-36).

As illustrated in Figure 1, folate-

dependent methionine cycle modulates

HCY availability for the transsulfuration

into cysteine. Homocysteine (HCY) is

converted to methionine by methionine

synthase enzyme (MS), which utilizes

vitamin B12 as a cofactor and acquires a

methyl group from, 5-

methyltetrahydrofolate (5-CH3-THF)

which is then converted to

tetrahydrofolate (THF). Methionine is

further converted to S-

Adenosylmethionine (SAM) through the

activity of methionine

adenosyltransferase. SAM is the major

methyl donor for all methyltransferases

enzymes, which add methyl groups to

various acceptor molecules such as

DNA, RNA, phospholipids and proteins.

SAM is then converted to S-

Adenosylhomocysteine (SAH), which is

then reversibly converted to HCY in a

reaction catalyzed by SAH-hydrolase.

HCY is either re-methylated back to

methionine or transsulfurated to

glutathione (GSH), the major cellular

antioxidant.



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Figure 1: Simplified schematic diagram of the folate-dependent HCY methylation

and transsulfuration pathway

Folate-dependent transulfuration

pathway is the precursor for de-novo

synthesis of reduced glutathione (GSH),

a tripeptide of glutamic acid, cysteine,

and glycine. GSH is the major

intracellular antioxidant and it undergoes

oxidation to the disulfide form (GSSG),

oxidized form, when scavenging reactive

oxygen species (ROS) that are highly

unstable molecules. In healthy cells and

tissues, more than 90% of the total

glutathione pool is in the reduced form

(GSH), and less than 10% exists in the

disulfide form (GSSG), and a reduced

GSH/GSSG ratio is an indication of

oxidative stress (37, 38). Oxidative

stress is a condition under which the

intracellular antioxidant (GSH),

antioxidant enzymes (glutathione

peroxidase, superoxide dismutase, and

catalase), and dietary antioxidants

(vitamin C, selenium, β carotene and

vitamin E) are not counterbalancing the

ROS, and subsequently induce cellular

damage which is involved in the

pathogenesis of cancer. Vegetables and

fruits are good sources of antioxidants

that provide a protective effect against

the ROS-mediated DNA and lipids cell

membrane structures damage in

proliferated cells (39, 40).

In conclusion various Western studies

have revealed that CRC carcinogenesis

is modifiable in accordance to certain

dietary factors with potential anticancer

properties. Such studies are needed to be

conducted in the Arabian Gulf region, in

order to address the intake of bioactive

food components in the diet consumed

by vulnerable groups at risk for CRC.

Addressing the physical activity is an

asset to develop a multidisciplinary

approach for the primary prevention of

CRC among high risk population. In the

Arabian Gulf region, primary prevention

of CRC is feasible through intervention

programs such as; establishing a

biochemical profile for early diagnosis

of CRC and adopting long-term behavior

modifications in food choices, dietary

pattern and physical activity.



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Should Children with Autism Continue Taking Folic Acid Supplement? Mostafa I. Waly,

1,2*

1Department of Food Science and Nutrition, College of Agricultural and Marine Sciences, Sultan

Qaboos University, Muscat, Oman 2Department of Nutrition, High Institute of Public Health,

Alexandria University, Egypt

*Corresponding Author: Dr. Mostafa Waly. Email: [email protected]

Dear Reader,

Elevated levels of homocysteine have been linked with autism. Despite evidence that

folic acid supplementation reduces the homocysteine level. There is a need for clinical

trials to specifically study the role of folic acid in the primary and secondary prevention

of autism. Sporadic evidence from childhood practices that there is a benefit for folic acid

supplementation for autistic children and those patients are encouraged to take folic acid

as a medication. Meta-analysis studies are needed to study the cause-specific effect of

folic acid deficiency and/or hyperhomocysteinemia on the autism incidence.

Waly MI. Should children with autism continue taking folic acid? Canad J Clin Nutr 2013;

1(1): 74.

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