1

Association between betel nut consumption and folate

deficiency among pregnant women in Matlab, Bangladesh

by

Manzur Kader

Master in International Health, 2011

IMCH, Department of Women‘s and Children‘s Health

Uppsala University, Uppsala, Sweden

Supervisor

Professor Lars Åke Persson Professor and Head of the Department, IMCH,

Department of Women‘s and Children‘s Health

Uppsala University, Sweden

June 14, 2011

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CONTENTS

Abstract……………………………………………………………...... 4

Introduction…………………………………………………………… 5-10

Aim……………………………………………………………………. 11

Materials and Methods ………………………………………………… 11-14

Study area……………………………………………………... 11

Design and study population…………………………………. 11-12

Ethical considerations…………………………………………. 12

Variables……………………………………………………… 12-13

Analysis………………………………………………………. 13-14

Results…………………………………………………………………. 14-19

Discussion……………………………………………………………. 19-22

Conclusion……………………………………………………………. 22-23

References…………………………………………………………….. 24-28

Annexes…………………………………………………………….. 29-35

Acknowledgments…………………………………………………….. 36

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LIST OF ABBREVIATIONS

BN Betel nut

AN

NGL

NGC

MNPN

ROS

DNA

GABA

SCE

p

IHD

HDSS

ICDDR, B

Areca nut

N-nitrosoguvacoline

N-nitrosoguvacine

3-methylnitrosaminopropionitrile

Reactive oxygen species

Deoxyribonucleic acid

Gamma-aminobutyric acid

Sister chromatid exchange

p-value

Ischemic heart disease

Health and demographic surveillance system

International Centre for Diarrheal Disease Research, Bangladesh

MINIMat

BMI

SD

ANCOVA

SPSS

OR

CI

Ca(OH)

Maternal and Infant Nutrition Intervention in Matlab

Body Mass Index

Standard deviations

Analyses of covariance

Statistical Package for the Social Sciences

Odds ratio

Confidence interval

Calcium hydroxide

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ABSTRACT

Background: Betel nut (BN) or Areca nut (AN) chewing habits on its own or with other ingredients

including chewing tobacco are highly prevalent in many South East Asian countries as well as among the

migrants from these countries residing around the world. The major alcaloid arecoline in betel nut has

been found to carcinogenic and to be associated with a range of health risks, including negative effects on

pregnancy outcomes. It is also reportedly associated with nutritional deficiencies including lower folate

status among men and women. Pregnancy imposes stress on folate stores because of increased

requirements for growth of maternal tissues, the fetus and the placenta. Folate deficiency during

pregnancy is a major public health concern as it is associated with many adverse health outcomes

including neural tube defects, low birth weight, preterm birth, delayed maturation of the nervous system,

growth retardation and megaloblastic anemia.

Aim: To evaluate the association between betel nut use and folate status among pregnant women in

Matlab, Bangladesh.

Design: A secondary analysis of data from the Maternal and Infant Nutrition Intervention in Matlab

(MINIMat) trial. A sub sample of pregnant women aged 14-50 years and living in the study area Matlab

with available serum folate measurements (N= 730) was included in this study. Outcome variable was

folate level and folate deficiency and the independent variable was betel nut consumption. Logistic

regression analysis and analyses of covariance (ANCOVA) were done. Results were adjusted for potential

confounders.

Results: Two-thirds (63%) of the women consumed betel nut and 17% had folate deficiency. Women

who consumed betel nut combined with chewing tobacco were 2.57 times more likely to have folate

deficiency (OR = 2.57; 95% CI = 1.23-5.36; p =0.012;) and betel nut consumption 2-3 times/day was

significantly associated with folate deficiency among users (OR = 2.51; 95% CI = 1.07–5.92; p =0.035).

Mean serum folate levels were significantly lower among betel nut users as compared to non-users.

Conclusion: Betel nut consumption combined with chewing tobacco is associated with lower serum

folate level or folate deficiency among pregnant women in Matlab in Bangladesh. Strategies are needed

for prevention and control of betel nut consumption in order to prevent adverse health outcomes. There is

a need of more knowledge regarding betel nut use and other combined substances to distinguish their

individual effects on folate deficiency.

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INTRODUCTION

Betel nut consumption: Betel nut (BN) or Areca nut (AN) is the seed of the areca palm (Areca

catechu), which grows on the Indian subcontinent and in South East Asia. Betel nut is mainly

consumed by chewing on its own or in combination with a range of other ingredients. Betel nut

consumption is a popular social habit and a ritual. Almost 15% of the world‘s population (600

million people) currently has betel nut chewing habits (1, 2). Betel nut use is popular in India,

Bangladesh, Sri Lanka, Myanmar, Maldives, Taiwan and few islands in the South Pacific. It is

also popular in many areas of Thailand, Malaysia, Indonesia, China, Philippines, Vietnam

Cambodia, Laos and among migrant populations from these countries residing around the world.

Betel nut is an important agricultural product in these countries (2, 3); (see Annex 1).

Betel nut or areca nut is sold or consumed as a food substance, although its food value is

uncertain. Ripe betel nut is orange-yellow in color and the consumable part is the seed

(endosperm), which is separated from the fibrous pericarp. There are varieties of ways to

consume betel nut. It can be consumed by itself as fresh or dried or it is processed by boiling,

baking or roasting before consumption. Fermented betel nut is also popular in some countries,

especially in Eastern India and southern Sri Lanka. In Taiwan, unripe green betel nut is often

consumed and has an appearance as a small olive (2, 4); (see Annex 2, 3).

The method of consumption or chewing can vary widely from country to country, but the most

commonly described form is the ‗‗quid‘‘ (synonymous with ‗pan‘ or ‗paan‘) or folded leaf

package which is made of a betel leaf, sliced betel nut and paste of lime (calcium hydroxide).

Smokeless or chewing tobacco is another important ingredient added to betel nut products

mainly in India and Southeast Asia. Tobacco is not usually added in Taiwan, Malaysia,

Cambodia, and China. Some other common ingredients are sweeteners, various spices, such as

cardamom, cloves, camphor, nutmegs, fennel etc. to suit individual taste and local custom. This

quid is placed in the mouth and chewed episodically to extract the juice (2, 3); (see Annex 2).

Due to very positive advertising strategies and aggressive campaigns, some betel nut products

have become very popular among all population groups, even among children in many south

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Asian counties and in migrant communities living in United Kingdom (UK). Pan masala, Gutkha

and Zarda are such type of products. Pan masala is basically an industrial betel nut product

containing cardamom, lime and a number of flavoring materials. Gutkha is a type of pan masala,

which also contain flavored chewing tobacco. Both products are often sweetened to enhance the

taste. Zarda is a type of flavored chewing tobacco, which is popular in many countries in South

Asia (2, 4, 5); (see Annex 2).

The consumption of betel nut has high addictive and abuse potential, as strong as cigarettes. So

its habitual and chronic daily use is well recognized in many cultures with almost 50% of users

consuming on a daily basis (2, 6). Even early initiation of betel nut use with tobacco products

among school children in Bangladeshi communities in UK is now a common situation (4, 6).

It is complicated to estimate quantities of the various ingredients of betel quid and prevalence

patterns of betel quid chewing. An ordinary quid consumed in Bangladeshi communities may

contain one betel leaf (approx. 1 g dry weight), a betel nut (approx. 4g), paste of lime (approx.

0.4 g) and some form of chewing tobacco e.g. tobacco leaves or zarda (0.4 g ). The quantities

and ingredients in a quid can vary among users. Some chewers do not add tobacco and many

chewers add some other flavoring ingredients like sweetened pan masala, coconut powder and

cumin (7); (see Annex 2).

The reported prevalence of betel quid chewing varies across Asian countries; India (Bombay):

34.5% among men, 27.2% among women, Thailand: 16% men, 19% women, Taiwan: 28.2%

men, 1.4% women (4) and Cambodia: 6.8% men, 40.6% women (1, 2). The prevalence is also

very high among many migrant populations living in UK. The major migrant communities

include those originating from Bangladesh, Pakistan, Sri Lanka and India (mainly Gujarat). The

prevalence of betel quid chewing has been estimated in British Bangladeshi from 78% to 96%

and in British mixed Asian groups from 27% to 47% (8).

There is an increasing trend of betel quid use among women in Bangladesh. Of all types of

tobacco products used, 47% was betel quid and 25% was dried tobacco consumption among

women when investigated on the national level (9). People with lower socio-economic status,

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women, older people, smokers, less educated and urban people are more likely to chew the betel

nut (8, 10).

Betel-quid chewing has been claimed to produce a sense of well-being, sweeten the breath,

dispel nausea, remove intestinal helminthes, strengthen gum, satisfy hunger, promote digestion,

reduce depression, etc. Women claim that betel quid chewing in the first trimester of pregnancy

can give relief from pregnancy-related nausea (3, 11). Information is scarce, however, regarding

effects of betel nut use on pregnancy outcomes and nutritional status.

Harmful effects of betel nut/quid: Betel nut contains 5-40% of different polyphenols as well as

many types of alkaloids such as arecoline, arecaidine, guavacoline and guavacine. The main

alkaloid is Arecoline, present up to 1% of its dry weight and other alkaloids are very small in

amount (5, 12). Many carcinogenic properties of betel nut have been identified in a large number

of experimental studies (4-6, 13, 14); (see Annex 4, 5, 6).

Several carcinogens are derived from betel nut alkaloids and commonly documented carcinogens

are nitrosamine products. Nitrosamines are produced by nitrosation of the alkaloid in dried stored

nuts, in the mouth and especially in the acidic condition of stomach. The main three betel nut

specific nitrosamines are N-nitrosoguvacoline (NGL), N-nitrosoguvacine (NGC) and 3-

methylnitrosaminopropionitrile (MNPN) that were detected in the saliva of betel quid chewers

with or without tobacco. Although betel nut specific nitrosamines have been well documented as

oral carcinogens, but the favorable acidic pH condition of stomach can induce nitrosation of

secondary and tertiary amines among consumers. It is even 2-3 time more carcinogenic with

ingested tobacco in the stomach (4, 13, 14). Thus, the betel quid chewer who swallows the quid

can be further exposed to these endogenous nitrosamines, which can lead to metabolic diseases

of other organs via gastrointestinal absorption.

Several polyphenols in betel nut have been shown to be genotoxic probably by formation of

reactive oxygen species (ROS) in the chewer's saliva. ROS causes oxidative stress or oxidative

degradation where the cell is unable to counterbalance. ROS can cause many harmful effects like

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oxidative and chromosomal damage of DNA, which could be involved in several stages of the

carcinogenic process in oral mucosa (5, 15).

Human studies have found that arecoline affect both sympathetic and parasympathetic nervous

systems. Arecoline appears to have a stimulating effect upon the sympathetic system with

elevation of adrenaline and noradrenaline levels. However, it also stimulates the parasympathetic

activities characterized by dilation of the pupil, increase in skin temperature, pulse rate and

systolic blood pressure. These effects on the autonomic nervous system possibly occur in a dose-

dependent manner where a higher dose stimulates the parasympathetic activity (6, 14, 16). Some

alkaloids have anti-muscarinic activities on smooth muscles. They can bind to GABA receptors

in the brain and act as inhibitors to GABA receptors and pose extensive effects on the brain,

pancreas, cardiovascular system, lungs and gastrointestinal tract (14, 16). Although chewing

leads to gastrointestinal absorption when it is swallowed, the primary route is direct systemic

absorption through buccal and sublingual mucosa that leads to an onset of effects within 5

minutes and can last for 2-3 hours among consumers. In some rare cases this can cause serious

physical problems such as cholinergic crisis, asthma attack, psychosis and cardiac arrhythmias or

aggravating cardiac diseases in vulnerable patients. (17, 18).

Harmful effects of betel nut have been observed in experimental studies on human embryos and

pregnant women (19-21). There is evidence that the alkaloid arecoline in betel nut can be found

in the neonatal biological matrices of infants born to mothers, who were exposed to arecoline

during pregnancy. In addition, its cytotoxicity and carcinogenic effects have been recognized and

found in meconium, cord sample and urine (19). Arecoline has also been found to relax human

umbilical vessels in a dose-dependent manner. In addition, arecoline has been found to inhibit

endothelial cell proliferation and arrest cell cycle in a dose-dependent manner (20). This is

further supported by a study of betel nut on DNA damage by inducing sister chromatid exchange

(SCE). The betel nut induces SCE more in pregnant women than in normal women (21). Thus,

betel nut is likely to produce adverse pregnancy outcomes among pregnant women by inducing

SCE and increasing DNA susceptibility to damage.

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Arecoline in betel nut may directly stimulate the central nervous system. This process includes a

rapid crossing of the blood–brain barrier, acceleration of heart rate, increasing of blood flow in

carotid arteries, but decrease in diastolic blood pressure via a peripheral cholinergic effect (22).

Accordingly, this might reduce maternal–fetal blood flow, which may have a negative effect on

birth weight.

Studies were conducted by Gamble et al. (23) and Pilsner et al (24) using data from a cohort

study among 973 women and 677 men between September 2000 and May 2002 in Bangladesh,

and showed that betel nut had an independent adverse effect on folate status. Gamble et. al. (23)

revealed that the plasma folate concentrations were lower for betel nut users than for nonusers

(9.9±6.1 and 12.0±7.7 nmol/L, respectively, p<0.0001) and cigarette smokers than for

nonsmokers (9.3 ±5.5 and 12.3 ±7.8 nmol/L, respectively; P < 0.0001). Pilsner et al. (24) found

that the participants in the lower folate group were more likely to be male, smoke cigarettes, and

to chew betel nut. Adjustment was made for age and sex in the study by Gamble et al. (23) and

pregnancy and vitamin B12 deficiency were exclusion criteria for the study by Pilsner et al. (24).

Other studies found that use of betel nut/quid was associated with thiamine deficiency (25, 26),

aggravated effects of vitamin-D deficiency (27), and was associated with central obesity,

hyperglycemia, diabetes mellitus (14, 28), cirrhosis (29) and goiter (30).

Various studies have showed that betel nut use during pregnancy has adverse effect on birth

outcomes, including low birth weight, shorter birth length and preterm delivery (31, 32). A

recent study from eastern and southern Taiwan among aborigines found the prevalence of betel

quid consumption during gestation to be 36.7%. Two-thirds of aboriginal women chewed betel

quid and a majority (86%) continued their habit during pregnancy (31). Only 45% of the

participants were aware of harmful effects of betel quid (32). In Mumbai, India (17.1%) of

pregnant women reported to have used betel quid with tobacco regularly during pregnancy (33).

Folate and pregnancy: Folate or folic acid is a water-soluble vitamin of the B-complex group

and it plays an important role for optimal health, growth and development. Folate is found

naturally in food and folic acid is the synthetic form of folate. Folate acts as cofactor or co-

enzyme and is involved in a variety of physiological processes such as amino-acid metabolism,

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maintenance and repair of genome, regulation of gene expression, neurotransmitter synthesis,

and the formation of myelin (34). Folate deficiency is a prevalent vitamin deficiency throughout

the world due to variety of reasons including impaired metabolism in some conditions such as

diseases of the proximal small intestinal, chronic use of certain drugs; e.g. anticonvulsants and

oral contraceptives, alcohol, renal and liver failure etc. Inadequate intake of folate-containing

foods (e.g. green leafy vegetables, citrus fruits) prolonged cooking, vitamin B12 (cobalamin), C

and Zinc deficiency and genetic defects can also cause folate deficiency (35-38).

Pregnancy imposes stress on folate stores because of increased requirements for growth of

maternal tissues, the fetus and the placenta. There is demand for folate for uterine enlargement

and for rise in blood volume, which requires increased red cell production. Increased folate

requirements during infancy, lactation, concurrent infection and malignancy may lead to folate

deficiency. Folate deficiency during pregnancy is a major public health concern as it is

associated with many adverse outcomes including increased risk for neural tube defects, low

birth weight, preterm birth, delayed maturation of the nervous system, growth retardation and

megaloblastic anemia (35, 36).

A number of clinical studies have demonstrated that folate deficiency is associated with mild-to-

moderate hyperhomocystinemia that could lead to atherosclerotic vascular disease such as

ischemic heart disease (IHD) and stroke (37-39).

A high prevalence of folate deficiency among Bangladeshi men and women has been

documented in many studies (23, 40-42). The prevalence of deficiency varies from 13% to 25%

among adolescent girls and women in Bangladesh (40-42).

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AIM

The aim of this study is to evaluate the association between of betel nut use, and form of betel

quid, and folate status among pregnant women in Matlab, Bangladesh.

MATERIALS AND METHOD

Study area: This study was conducted using secondary analysis of data from a randomized

control trial in Matlab, Bangladesh. Matlab is subdivision in Bangladesh, about 55 kilometers

southeast of Dhaka, the capital of Bangladesh (see Annex 7). Matlab is a major rural field site

and a reputed public health resource for the world. This site represents a model for public health

strategies around the world. Matlab has been under health and demographic surveillance system

(HDSS) of the International Centre for Diarrheal Disease Research, Bangladesh (ICDDR, B)

since 1966 and this HDSS covers about 225,000 population living in Matlab area and provides

necessary socio-demographic data to plan, conduct, and evaluate various types of public-health

interventions. The main source of income in the area is agriculture, but fishing is the means of

livelihood in a few villages. The majority of the farmers possess less than 2 acres of land and

40% are landless. Therefore, many households live on sharecropping and farming others‘ land on

a daily wage basis. However, some people work in other places e.g. in mills and factories but

their families live in the study area. The majority of the population is Muslim. The literacy rate is

low, particularly among older women (43, 44).

Study design and study population:

The data for this study were received from the Maternal and Infant Nutritional Interventions in

Matlab (MINIMat) trial, Bangladesh. MINIMat is a large food and multiple micronutrient

supplementation trial in pregnant women, including long-term follow-up of their children, and of

the mothers into the next pregnancy. A total of 4436 women living in Matlab, Bangladesh, were

invited and randomized for different interventions. Pregnant women (aged between 14-50 years)

who lived in the study area (Matlab) were invited to this trial once their pregnancy was

confirmed with a urine test and an ultrasound showing gestational age less than 14 weeks. A

random subset of 730 women representative of the full year 2002 was selected for folate status

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assessment. So a total of 730 women with available serum folate measurements were included in

the analysis of this master thesis.

Ethical consideration:

Since this is a secondary analysis of anonymous data, no new verbal or written consent from the

participants was necessary. This analysis was in line with the intention of the original study,

which included an informed consent from all participants. The MINIMat trial was approved by

the Research Review Committee and the Ethical Review Committee of ICDDR,B.

Variables:

Folate and vitamin B-12 status:

The outcome variable is folate deficiency among women at week 14 of gestation. Women with

serum folate levels less than 6.8 nmol/L were defined as folate deficient. Serum folate levels

were also available as a continuous variable. Serum vitamin B-12 less than 150 pmol/L was

defined as vitamin B-12 deficiency.

Betel nut consumption:

Women were asked about their consumption of betel nut during their last pregnancy at the time

of their post-partum visit. If they had consumed betel nut, they were further asked if betel nut

was consumed as dry or fresh or with combination of other ingredients such as betel leaf, lime

and zarda (chewing tobacco). The types of betel nut consumption was recoded into these

alternatives: Non-user of betel nut (BN) or user of BN; Non-user of BN or BN (fresh or dry) with

chewing tobacco or BN (fresh or dry) without chewing tobacco; Non-user of BN or dry BN with

chewing tobacco or fresh BN with chewing tobacco or dry BN without chewing tobacco and

fresh BN without chewing tobacco.

Frequency of betel nut was recorded as no consumption, less than daily, 1 time/day, 2-3

times/day.

Women‘s age was re-coded into 3 age groups (14-24, 25-30 and >30). Parity was recoded into no

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child, 1 child, 2 children or more than 2 children. Body mass index (BMI) was computed, then

categorized according to WHO definition of underweight (BMI <18.5), normal weight (BMI

18.5-24.9), overweight (BMI 25-29.9) and obese (BMI of 30 or greater).

Socioeconomic status:

The socioeconomic status of the women was assessed by using a household asset score recoded

into quintiles expressing the wealth index as poor, lower middle, middle, upper middle and rich.

Women‘s literacy was classified as ability to read and write or illiterate.

Analysis:

Merging, cleaning of data and control of missing values were performed as the primary step of

data analysis. Serum folate measurement at week 14 was taken as outcome variable.

Serum folate measurements were available for 730 women; thus, all analyses refer to these

women.

Baseline descriptive characteristics are presented as number, percentage, means and standard

deviations (SDs).

Associations between exposure (betel nut use) or potential confounding variables and

dichotomous outcomes were analyzed by use of Pearson‘s chi square test.

Multivariate analyses were used to evaluate the independent association of the identified factors

of importance for folate deficiency. Firstly, unadjusted odds ratio (OR) was obtained from binary

logistic regression models between outcome variable of folate deficiency and each independent

variable for types of betel quid consumption or various frequency of betel quid consumption.

Thereafter, potential confounders were added to the models one by one and were retained in the

final model if they changed a significant beta coefficient of the variables of key interest

with >10%. Identified potential confounders were calculated asset score, women‘s age, serum

vitamin B-12 status and women‘s literacy.

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Analyses of covariance (ANCOVA) models were performed to analyze whether folate levels

were associated with betel quid consumption. Other covariates, i.e. asset score, age, serum

vitamin B-12 status and literacy that could influence the dependent variable were added to the

model to get adjusted models with mean, p-value and 95% confidence interval.

All statistical analyses were performed by using IBM SPSS Statistics 19 for Window (SPSS Inc,

Chicago, IL). Results with p-value of <0.05 were considered to be statistically significant

RESULTS

Basic characteristics:

The basic characteristics of the women are presented in table 1. On average the women were 26

years old, 45 kg in weight and 150 cm in height. The mean BMI was 20 kg /m2 and mean parity

was 1.4. Around two-thirds (63%) of women could read and write in the study area.

Betel nut consumption: Almost two-thirds (61%) of women consumed betel nut. A majority of

women (58%) consumed dry betel nut and only 3.6% consumed fresh betel nut. Half (52%) of

women consumed betel nut with ingredients betel leaf and lime but without chewing tobacco and

9.8 % of women added chewing tobacco with it. Regarding frequency of consumption, 10.7%

women consumed betel nut 2-3 times per day, 13.4 % consumed once per day and 5.6%

consumed less than daily.

Folate deficiency: Of the 730 women included in this analysis, 124 (17%) had folate deficiency.

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Table 1: Baseline characteristics of participating women

Variables N Mean ± SD

Age 730 26.6 ± 5.9

Weight 726 45.3 ± 6.9

Height 730 149.9 ± 5.4

BMI 726 20.1 ± 2.7

Parity 730 1.4 ± 1.4

N %

Asset score

Poor 130 17.8

Below middle 172 23.6

Middle 150 20.5

Upper middle 130 17.8

Rich 148 20.3

Literacy

Illiterate 245 33.6

Can read and write 485 66.4

Betel nut (BN) consumption

Non user of BN 238 38.8

Betel nut user 376 61.2

BN without chewing tobacco 317 51.6

BN with chewing tobacco 60 9.8

Dry betel nut 299 57.7

Fresh betel nut 18 3.6

Folate status

Non-deficient 606 83

Deficient 124 17

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Associations between independent variables and outcome folate deficiency are shown in

table 2. Of the variables listed in the table, only vitamin B-12 deficiency was significantly

associated with folate deficiency.

Table 2: Associations between independent variables and folate deficiency of the women

Folate deficient Non-deficient

Variables All N (%) N (%) N (%) p-value† Asset Poor

Score Below middle

Middle

Upper middle

Rich

Total

130 (17.8) 100 (13.7) 30 (4.1)

172 (23.6) 141(19.3) 31 (4.2)

150 (20.5) 127 (17.4) 23 (3.2)

130 (17.8) 113 (15.5) 17 (2.3)

148(20.3) 125 (17.1) 23 (3.2)

730 (100) 606 (83.0) 124 (17.0) 0.242

Age (year) 14-24

25-30

>30

Total

277 (39.9) 226 (32.5) 51 (7.3)

207 (29.8) 165 (23.7) 42 (6.0)

211 (30.4) 186 (26.8) 25 (3.6)

695 (100) 577 (83.0) 118 (17.0) 0.051

Parity No child

1 parity

2 parity

≥3 parity

Total

241 (33.0) 200 (27.4) 41 (5.6)

196 (26.8) 156 (21.4) 40 (5.5)

152 (20.8) 131(17.9) 21 (2.9)

141 (19.3) 119 (16.3) 22 (3.0)

730 (100) 606 (83.0) 124 (17.0) 0.407

BMI (kg/m2) Underweight

Normal weight

Overweight

Total

194 (26.9) 163 (22.6) 31(4.3)

487 (67.6) 400 (55.6) 87 (12.1)

39 (5.4) 33 (4.5) 6 (0.8)

720 (100) 596 (82.8) 124 (17.2) 0.801

Literacy Illiterate

Can read and write

Total

245 (33.6) 196 (26.8) 49 (6.7)

485 (66.4) 410 (56.2) 75 (10.3)

730 (100) 606 (83) 124 (17) 0.084

Vitamin B-12 status Normal

Deficient

Total

378 (53.9) 335 (47.8) 43 (6.1)

323 (46.1) 250 (35.7) 73 (10.4)

701 (100) 585 (83.5) 116 (16.5) >0.001

†Differences assessed with Pearson‘s chi-square tests

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When the background variables potentially associated with folate deficiency were included in

logistic regression models (table 3), women who consumed betel nut with zarda (chewing

tobacco) were 2.57 times more likely to have folate deficiency (OR = 2.57; 95% CI = 1.23-5.36;

p =0.012;). Age and vitamin B-12 deficiency were also significantly associated with folate

deficiency in this model.

If focusing the frequency of betel nut consumption it was found that betel nut use 2-3 times per

day (disregarding whether combined with tobacco or not) was significantly associated with folate

deficiency among users (OR = 2.51; 95% CI = 1.07–5.92; p =0.035 ;). Asset score and literacy

were not associated with folate deficiency.

The mean differences in folate levels among various types and frequency of betel nut consumers

are shown in table 4. The mean plasma folate levels were significantly lower for all of the betel

nut (BN) users as compared to nonusers (mean 11.52, and 13.08 nmol/L, respectively, p=0.006).

The mean plasma folate levels were lower for all of the betel nut users with and without chewing

tobacco in comparison with non-users (mean 10.53, 11.70 and 13.10 nmol/L, respectively,

p=0.009 and p= 0.017). The mean plasma folate levels were significantly lower for all of the dry

BN users with tobacco as compared to all of the fresh BN users with tobacco and non-users

(mean 11.49, 11.94 and 13.08 nmol/L, respectively, p=0.006).

The mean plasma folate levels were significantly lower for all of the dry BN users without

tobacco compare to all of the fresh BN user without tobacco and non-users (mean 11.63, 12.49

and 13.08 nmol/L, respectively, p=0.016). These results were significant after adjusting for

potential confounding factors, i.e. asset score, vitamin-12-deficiency and literacy.

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Table 3: Logistic regression for adjusted and unadjusted odds ratio (OR) of folate deficiency

Adjusted OR Adjusted OR

Unadjusted OR

Predictors

Model a Model b

N OR CI (95%) OR CI (95%) OR CI (95%)

BN consumption

No consumption 237 Reference

Betel nut only 317 0.97 0.61-1.56 0.90 0.53-1.52

BN with chewing tobacco 60 2.39** 1.24-4.61 2.57* 1.23-5.36

Frequency of consumption

No consumption 262 Reference

Less than daily 21 0.87 0.24-3.00 0.62 0.13-2.86

Once daily 50 0.85 0.36-2.02 1.07 0.40-2.87

2-3 times daily 40 2.25* 1.06-4.76 2.51* 1.07-5.93

Women’s age

14-24 years of old 277 1.68* 1.00-2.81 2.27* 1.17-4.40 2.34 1.00-5.48

25-30 years of old 107 1.89* 1.11-3.24 2.56** 1.32-4.97 2.70* 1.16-6.27

> 30 years of old 211 Reference

Vitamin B-12 status

Normal Vitamin B-12 378 Reference

Vitamin B-12 deficiency 323 2.28* 1.51-3.43 2.45** 1.51-3.98 2.12* 1.14-3.91

* Significant at p‐value <0.05; ** Significant at p‐value <0.01

Model a Adjusted for types of betel nut consumption, calculated asset score, women‘s age, vitamin B-12 status and

literacy

Model b Adjusted for frequency of betel nut consumption, calculated asset score, women‘s age, vitamin B-12 status

and literacy

19

Table 4: The mean folate levels among women using or not using betel nuts (types and

frequency). ANCOVA, unadjusted and adjusted models with mean, mean difference and p-value.

Unadjusted Adjusteda

Betel nut consumption N Mean folate

(nmol/L)

Mean

difference

Mean folate

(nmol/L)

Mean

difference

Betel nut (BN) users

Non user

376

238

11.41**

12.92

-1.51

Ref.

11.52**

13.08

-1.56

Ref.

BN with tobacco

BN without tobacco

No use

60

317

237

10.62*

11.55*

12.95

-0.93

Ref.

10.53**

11.70*

13.10

-1.17

Ref.

Dry BN with tobacco

Fresh BN with tobacco

No use

354

22

238

11.40**

11.61

12.92

-0.21

Ref

11.49**

11.94

13.08

-0.45

Ref

Dry BN without tobacco

Fresh BN without tobacco

No use

299

18

237

11.50*

12.30

12.95

-0.80

Ref

11.64*

12.49

13.08

-0.85

Ref

a Adjusted for asset score, age, vitamin B-12 deficiency and literacy. No betel nut use is Reference category.

* Significant at p‐value <0.05; ** Significant at p‐value <0.01

DISCUSSION

We have shown that betel nut consumption with chewing tobacco was associated with lower

serum folate level and folate deficiency in pregnant women in Bangladesh.

According to ANCOVA, the betel nut without chewing tobacco was associated on lower folate

status and in logistic regression, betel nut with chewing tobacco was associated with folate

deficiency.

To our knowledge this is the first study which shows that the betel nut consumption with

chewing tobacco is significantly associated with folate deficiency amongst its consumers. Two

previous studies documented that the betel nut use was associated with lower plasma folate

levels (23, 24).

20

The results also suggest that in addition to betel nut and chewing tobacco, vitamin B-12

deficiency is an important determinant of folate deficiency and women in fertile age (14-30

years) are vulnerable to folate deficiency. It is very well documented that vitamin B-12

deficiency is a major cause of folate deficiency (35-38). Folate deficiency among fertile women

was relatively common in this study, which was consistent with previous studies where women

below 40 years of age in Bangladesh (40) and women between 18 and 22 years of age in India

(45) were folate deficient, 17% and 26.3% respectively.

In this study 9.8 % of women added chewing tobacco in the betel quid, but women in

Bangladeshi community living in UK reported to add more chewing tobacco, ranges from Tower

Hamlets 43% (46), Yorkshire 69% to Birmingham 81% in their quids (4). Thus, these British

women with Bangladeshi background may also be more exposed to this risk factor.

Betel quid consumption 2-3 times per day had a significant association with folate deficiency.

However, other studies calculated that the Bangladeshi communities in UK consume an average

of 6 quids per day (7, 47). Even if the UK Bangladeshi women cannot be compared with rural

poor women in the Bangladesh delta, the dose-response relationship suggested in our study

indicates that even betel nut use in Europe may be associated with folate deficiency problems.

Almost 17% of these women had folate deficiency. The high prevalence of folate deficiency in

early pregnancy is a major concern, as it could lead to adverse pregnancy outcomes and

increased health risks for both mother and child. This study has the advantages of randomized

enrolment of pregnant women over a full year of 2000 that made the results representative of the

study population and hence the findings can be generalized to pregnant women in the area and

most likely in common segments of Bangladesh. In the census of Matlab HDSS study area in

2005, the socio-economic characteristics of this area were similar to that of national level (43).

Fresh betel nut use was not significantly associated with lower folate levels . A possible

explanation for this may be due to a relatively small sample size of women using fresh betel nut.

Dry betel nut is the main ingredient of betel quid in this study as well as other studies (4, 31, 32).

21

It is notable that two important potential confounders alcohol and cigarette smoking were absent

among the women in this study.

Poor populations are usually vulnerable to nutritional deficiencies. The lack of association

between socioeconomic status and folate deficiency in this study may be related to the fact that

the entire population is relatively poor.

This study was a cross-sectional study to investigate the association between betel nut exposure

and folate status. Such a design does not give any indication of the sequence of events. This

precludes possibilities to draw causal inferences from the results.

It was not possible to disentangle the association between other ingredients such as lime and

betel leaf (pan) used with betel nut and the outcome. There was no previous evidence of any

association of these ingredients with nutritional deficiency. Some studies have reported that the

use of lime or Ca(OH)2 by betel-quid chewers play an important role in the generation of ROS

which is responsible for development of several stages of oral cancer (15). On the other hand,

betel leaf or Pan contains large amount of anti-oxidant carotenes (80.5 mg/g), which are known

to reduce the risk of cancer. Betel leaf extracts act as effective scavengers of ROS. Betel leaves

also contain several compounds such as chlorophyll, chavicol and hydroxychavicol, which are

thought to be have chemoprotective properties (4, 5, 14).

Another limitation of the study was the unavailability of data on dietary intake of folic acid and

vitamin B-12. A recent food base study reported that vegetables, fish, pulses and rice are the key

daily dietary items of the population in this study area. Some of the vegetables include amaranth

(Amaranthus viridis), spinach (Basella alba), bitter gourd (Momordica charantia), arum

(Colocasia esculenta) stem, green banana (Musa spp.) and eggplant (Solanum melongena) (48).

A majority of these food items are rich in folate, thus the evidence suggests that the dietary folate

intake is not inadequate among the women in this study setting. But eating a mainly vegetarian

diet can be a cause of vitamin B-12 deficiency among the women.

22

Earlier published data are insufficient to provide a clear picture of the prevalence pattern of betel

nut or quid chewing by Bangladeshi populations. Some of the reports reflect the habits of

Bangladeshi migrant populations living in the UK. It is clear that the habit of betel quid chewing

with and without tobacco is widespread among Bangladeshi population and therefore an

indication of a similar pattern of the chewing habits in their home country.

CONCLUSION

The results of this cross-sectional data analysis show that betel nut use is significantly associated

with lower folate status among pregnant women and addition of chewing tobacco is associated

with folate deficiency.

Although this study focused on pregnant women, lower folate status or folate deficiency is also

likely in the general population, who are consumers of betel nut. A majority of the population in

South East Asian countries may be unaware of harmful effects of betel nut and continue to

consume betel nut during pregnancy. The betel nut or betel quid chewing habits are widespread

and very well socially accepted in many South East Asian countries as well as amongst the

migrant populations from these countries. Social integrity with betel nut has been described as

―Betel is a symbolic offering on occasions of welcoming guests and paying homage, courtship

and betrothal, funerals and ancestral remembrances, reconciliation and peacemaking, and in the

practice of traditional medicine‖ (49).

Today, betel chewing products with or without tobacco are very popular among all age groups in

many South East Asian countries. And these products are shown as a safer alternative to tobacco

products by aggressive marketing campaigns, which also target children and adolescents. It is

important to educate individuals about the harmful effects of betel nut. If the population is not

educated the consumption of betel nut may continue to future generations as part of cultural

traditions. For an example, there was no difference found between the chewing habits of first-

and second-generation Bangladeshi migrations in the UK (1, 8).

Strategies to prevent and control betel nut consumption are needed in order to prevent adverse

health outcomes. An effective health promotion policy aimed at reducing the use of betel quid

23

may reduce its use. There is need for more precise data on various ingredients of betel nut to

distinguish their individual effects on folate deficiency.

24

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29

ANNEXES:

Annex 1: Production of betel nut by country since 1961 (in millions of tonnes), from FAO

(2003)

Country 1961 1971 1981 1991 2001

Bangladesh 62 995 23 369 25051 24120 47 000

India 120 000 141 000 195 900 238 500 330 000

Indonesia 13 000 15 000 18 000 22 812 36 200

Kenya NA NA NA 100 90

Malaysia 6 500 3000 2 500 4000 2500

Maldives 1 1 5 16 37

Myanmar 8000 19 203 25 807 32 270 51 463

Taiwan, China* 3718 10 075 24 358 111 090 165 076

Thailand NA NA NA 13 250 20 500

World 428 428 423 296 583 242 892 316 1 305 732

NA, not available, * From Council of Agriculture, ROC (2003)

Ref: IARC Monographs- Vol. 85

Annex 2: Betel nut and betel quid:

Betel nut: The seed or the endosperm of tree areca palm (Areca catechu). Betel nut is the main

ingredient of a variety of widely used chewed products. There are varieties of ways to consume

betel nut. It is mainly consumed by chewing on its own or in combination with a range of other

ingredients listed below:

30

Betel quid ingredients:

Fig: Betel nut or areca nut Fig: A betel quid prepared in the traditional way using

betel leaves, sliced areca nut, dry tobacco and lime paste

Fig: paste of lime Fig: A. betel quid ingredients

B. Some industrial products - pan masala and gutka

31

Betel quid: Quid (synonymous with ‗pan‘ or ‗paan‘) or folded leaf package which is placed in

the mouth and chewed episodically to extract the juice, some betel-quid products in many

countries are not actually chewed; instead, they are applied to the oral cavity and remain in

contact with the oral mucosa. Betel quid can be made of:

Constituent preparation

Betel nut or areca nut, ―Supari‖ in Bengali and

Hindi

Unripe/ripe

Whole/sliced

Raw/roasted/sun dried, boiled/soaked in water

Fermented (under mud)

Betel leaf or Piper betel leaf (pan) Fresh leaf , inflorescence, Stem

Paste of lime (calcium hydroxide)

From coral, from shell fish

From quarried lime stone

Tobacco Sun dried, boiled with molasses

Fermented, perfumed

Concentrated extract (kiwam)

Catechu Extract of Acacia catechu

Extract of Acacia suma

Spices, Sweeteners and essences Cloves, Cardamom, camphor, Aniseed,

Menthol Coconut Dried dates, nutmegs, fennel,

Mint, Rose petals

Ref: IARC Monographs- Vol. 85

32

Annex 3: Betel nut tree and fruits and flowers:

Fig: betel nut fruits hanging in a tree Fig: A diagrammatic illustration of areca palm

33

Fig: Unripe nut mainly consumed in Taiwan. Fig: florescence (flower) of Piper betle

mostly used in Taiwan In and Melanesia

Annex 4: Concentrations of the chemical constituents (Percentage based on dry weight) of a

variety of unprocessed green and ripe betel

nuts

Constituents Green (unripe) nut (%) Ripe nut (%)

Moisture content

Total polysaccharides

Crude protein

Fat

Crude fibre

Polyphenols

Arecoline

Ash

69.4–74.1

17.3–23.0

6.7–9.4

8.1–12.0

8.2–9.8

17.2–29.8

0.11–0.14

1.2–2.5

38.9–56.7

17.8–25.7

6.2–7.5

9.5–15.1

11.4–15.4

11.1–17.8

0.12–0.24

1.1–1.5

34

Annex 5: Alkaloid content (Percentage not specified, probably based on dry weight) of fresh

betel nuts from Darwin, Australia

Alkaloid %

Arecoline

Arecaidine

Guvacoline

Guvacine

0.30–0.63

0.31–0.66

0.03–0.06

0.19–0.72

Annex 6: Carcinogenic tobacco- and betel nu nitrosamines (range, ng/mL) detected in saliva of

chewers of betel quid with tobacco (BQ + T) and without tobacco (BQ)

Carcinogenic agent BQ + T (range, ng/mL) BQ (range, ng/mL)

Tobacco-specific- nitrosamine

N-nitrosonornicotine (NNN)

4-(methylnitrosamino)-

1-(3-pyridyl)-1-butanone (NNK)

N-nitrosoanabasine (NAB)

Areca nut-specific- nitrosamine

N-nitrosoguvacoline (NGL)

N-nitrosoguvacine (NGC)

3-(methylnitrosamino)propio nitrile (MNPN)

1.2–38

1–2.3

0–40

4.3–350

0–30.4 (6)

NR

ND

ND

NR

2.2–9.5

0–26.6

0.5–11.4

Ref: IARC Monographs- Vol. 85

35

Annex 7: Study area Matlab, a subdivision of Bangladesh, about 55 kilometers southeast of

Dhaka, the capital of Bangladesh

36

ACKNOWLEDGEMNT

I would like to express my deepest gratitude to my supervisor, Professor Lars Åke Persson. I

could not have imagined having a better supervisor for my thesis work and without his

invaluable guidance and support with insightfulness I would never have finished.

I am very grateful to Eva-Charlotte Ekström for being my examiner and allowing me to access

the secondary data. It would not be possible to conduct this study without her support and

encouragement from the beginning.

I am really grateful to Suman Kanti Chowdhary for his timely support with statistical data

analysis.

My special thanks to my friend Nirmala Perera for spending her precious time reading my paper

and giving me valuable comments and suggestions.

I like to extend my thanks to my group members: Dr. Guru Prasad A and Navan Tripati for

their valuable comments and questions during the preparatory sessions. On a different note, I

would extend my gratitude to all students and staffs of IMCH for their continued kind support.

I wish to express my greatest love and gratitude to my wife, my soul mate Saira Naim for her

understanding and endless love for me.

Last but not least, I am grateful for the most wonderful people in my life- my mother Abida

Sultana and my father Iyakub Ullah, for their love, support and encouragement throughout my

entire life and who taught me the value of a good education through their own struggles and

sacrifices.