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Effect of chewing betel nut on measurements of salivary progesterone and estradiol.

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Effect of Chewing Betel Nut on Measurements
of Salivary Progesterone and Estradiol
Alejandra Núñez-de la Mora,1 Robert T. Chatterton,2 Esnar T. Mateo,2 Fahmida Jesmin,1
and Gillian R. Bentley1*
Department of Anthropology, University College London, London WC1H 0BW, UK
Department of Obstetrics and Gynecology, Northwestern University, Chicago, IL 60611
saliva; radioimmunoassay; steroids; betel quid; Areca catechu
The measurement of steroids in saliva is
both simple and non-invasive and has been widely used
in field and clinical-based research. The observance of
particular cultural practices by some populations, however, may hamper accurate hormonal analyses. The present study evaluated the effects of one such practice—the
chewing of betel nut—on the accurate measurement of
salivary progesterone and estradiol. A time series experiment was conducted among Bangladeshi women who are
regular users of betel nut. Salivary steroids were ana-
The development of salivary assays for reproductive steroids has expanded the scope of sampling regimes in endocrinology as well as field-based research in reproductive
ecology. This non-invasive, simple way to obtain whole
menstrual cycle profiles of progesterone and estradiol has
revolutionized the study of the factors that impact ovarian
function (Riadfahmy et al., 1987; Ellison and Lipson,
1999) and has allowed comparisons among populations living in a variety of ecological settings (Ellison, 1994). Furthermore, the ease with which salivary samples can be collected makes this technique an excellent alternative for
the measurement of steroids in clinical research (Haeckel
and Hanecke, 1993; Voss, 1999; Lac, 2001). Many populations, however, observe cultural practices such as betel
nut, qat, tobacco or coca-leaf chewing, which could potentially hamper accurate hormonal analyses using saliva
(e.g., Vitzthum et al., 1993; Campbell et al., 2003).
Betel nut chewing is endemic in the Indian sub-continent, Southeast Asia, and large parts of the western Pacific as well as among migrants from these areas to other
countries (Summers et al., 1994; Warnakulasuriya, 2002).
It has been estimated that 10–20% of the world population
engage in this habit (Boucher and Mannan, 2002; Strickland, 2002). Betel nut is claimed to be a mild stimulant
and addictive agent and in different cultures it is regarded
as a general tonic for its physiological and psychological
effects (Burton and Burton-Bradley, 1979).
Betel chewing has been practiced for centuries and is
firmly rooted in the social, cultural, and economic life of
many populations (Strickland, 2002). In some regions, however, perceptions are beginning to change, and people with
colored teeth, resulting from heavy betel chewing are now
more likely to be deprecated. The practice of chewing betel
nut, nevertheless, is still widely regarded as socially acceptable, and in most countries is supported by positive marketing strategies as well as legal distribution (Croucher and
Islam, 2002; Gupta and Warnakulasuriya, 2002).
C 2006
lyzed by radioimmunoassay in samples collected prior to
and then 30, 60, 120, and 240 min following betel quid
use. Results show no significant difference between basal
steroid levels and those obtained 60, 120, and 240 min
after chewing betel nut. We conclude that with specific collection protocols that take into account time since chewing, salivary steroid analyses can be undertaken in populations among whom the practice of chewing betel nut is
endemic. Am J Phys Anthropol 132:311–315, 2007. V 2006
Wiley-Liss, Inc.
While the constituents of betel nut chewing may be consumed separately or in various combinations, the most
commonly described form is the \quid." The ingredients in
betel quid (also known as paan for its name in Hindi) vary
depending on geography, local custom, and personal preference, but the major components are betel nut (Areca catechu), betel leaf (Piper betel), slaked lime (calcium carbonate) as an alkaloid enhancer, and \catechu" an astringent
extract of the wood Areca catechu. Smokeless tobacco leaf
(either Nicotina tobaccum or Nicotina rustica) is a common additive along with other spices and flavorings such
as aniseed, fennel, and coriander seed (Williams, 1995;
Bedi, 1996; Strickland, 2002). The quid can be made up
personally or purchased ready-made. Generally, lime and
catechu are smeared on a betel leaf, which is then partially
folded into a funnel shape into which the small pieces of
betel nut and the rest of the ingredients are added. Once
folded completely, the resulting quid is placed in the
mouth—usually in the cheek—and gently chewed and
sucked. The quid may be stored in the buccal mucosa for
varying periods of time, ranging from 5 to 60 min, or even
overnight. Chewing encourages salivation, and the practice produces red saliva which discolors the teeth and lips.
Grant sponsors: Mexican National Council for Science and Technology–CONACyT (Mexico); CVCP Universities (UK) Postgraduate
Scholarships; Royal Society (UK).
*Correspondence to: Gillian R. Bentley, Department of Anthropology, Durham University, 43 Old Elvet, Durham DHI 3HN, UK.
Received 15 February 2006; accepted 7 September 2006
DOI 10.1002/ajpa.20513
Published online 28 November 2006 in Wiley InterScience
After chewing, the quid may be swallowed, or more often,
saliva and quid are expectorated.
Given the widespread nature of betel nut chewing in
South Asian and Pacific regions as well as among migrant
groups from these areas in to the West, and the increasing interest in the use of biomarkers for the study of
human variation among many of these populations, the
evaluation of research protocols is of critical importance.
Previous studies have reported that some stimulants
such as coca leaf and tobacco chewing have a significant
acute effect on salivary hormone values (Vitzthum et al.,
1993; Campbell et al., 2003). Although transitory, the significant rise in steroid levels following chewing of these
substances can lead to equivocal hormonal results unless
sample collection protocols are appropriately adjusted.
Such evidence suggests a similar potential contaminating
effect from betel nut chewing.
The aim of this study is to evaluate the effect of betel
chewing on the accurate measurement of salivary steroids using radioimmunoassay (RIA) and to assess the
feasibility of using this technique in betel nut chewing
populations. Specifically, we test the hypothesis that betel
nut chewing can have a significant but transitory effect
on salivary progesterone and estradiol levels.
Study population
Twenty Bangladeshi women living in London, UK, aged
18–42 and regular users of betel quid were recruited into
the study. Women were contacted through bilingual coordinators at local schools and community centers and
invited to participate in the study. After obtaining written
informed consent, women were screened for steroidcontraceptive use, menstrual cycle irregularities, infertility, diabetes, and thyroid disorders that might affect their
steroid hormone profiles. On the day of the saliva collection, a 15 min questionnaire on betel nut use, reproductive, medical, and migration histories was administered
by a bilingual (English-Sylheti) female research assistant.
Heights and weights were obtained following standard
anthropometric procedures (Gibson, 1990).
The research was approved by the East London and the
City Health Authority Research Ethics Committee, the
Camden and Islington Community Health Services NHS
Trust Local Research Ethics Committee, and the joint
UCL/UCLH Committee on the ethics of human research.
Data were collected and stored in compliance with the
Data Protection Act, UK.
Sample collection and analysis
To maximize potential levels of both progesterone and
estradiol, women were scheduled to participate in the
pilot study during the mid-luteal phase of their menstrual
cycle. Based on the date of their previous menses and on
the reported average length of their menstrual cycles,
participants took part in the study between 8 and 10 days
prior to the estimated date of their subsequent menstrual
period. Participants were followed up to obtain the exact
date of their subsequent menses, and to calculate retrospectively the actual luteal day on which the samples
were collected. Menstrual cycle days were then numbered
in reverse counting backwards from the subsequent first
day of menstruation, such that the last day of the menstrual cycle during which saliva was collected would be
counted as Day1. Samples outside the luteal phase
(<14 reverse menstrual day) were only assayed for estradiol.
Women were requested to refrain from chewing betel
nut in the evening preceding the study, and not to eat,
drink (except water) or brush their teeth (in case of bleeding gums which might contaminate the saliva sample) for
a minimum of 1 h prior to sample collection.
During the study, participants were asked to prepare
their customary betel quid using their own ingredients
and to chew as normal. It took them between 5 and
15 min to finish the betel quid. Some women spat out the
debris while others swallowed it.
Collection schedule
All saliva samples (5 ml) were collected in the morning
according to the following schedule:
Sample 1: Collected immediately prior to chewing the
betel quid (baseline levels).
Sample 2: Collected immediately after finishing chewing the betel quid.
Sample 3: Collected 30 min after sample 2.
Sample 4: Collected 60 min after sample 2.
Sample 5: Collected 120 min after sample 2.
Sample 6: Collected 240 min after sample 2.
Participants were allowed to drink water during the
study. Only three women consumed food (between samples 5 and 6), but finished eating on average 90 min prior
to Sample 6 collection.
Saliva was collected in polystyrene tubes pre-treated
with sodium azide to a final concentration of approximately 0.1% as a preservative. A sugarless spearmint flavored gum that has been extensively tested and determined not to interfere with analyses of salivary progesterone and estradiol (Lu et al., 1997) was used as a
salivary stimulant to replicate protocols that would normally be used in the collection of saliva samples from
non-chewing women.
Although betel nut chewing elicits profuse salivation,
its effect disappears after a few minutes after chewing
completion and thus an additional stimulant is required
in the collection of the pre-chewing and all later samples
in the series.
Saliva samples were stored and assayed using standardized radioimmunoassay techniques described previously
(Lu et al., 1997, 1999). Antiserum for the progesterone
assay has been characterized in a previous publication
(Chatterton et al., 1991). Antiserum and [125I]-estradiol
for the estradiol assay were obtained from Diagnostic
Services Laboratories, Webster, TX. All samples had comparable handling and treatment conditions and were
assayed in three batches within 2 months of collection.
Interassay variability averaged 39.9% for progesterone
and 34.1% for estradiol, and intraassay variability averaged 13.2% for progesterone and 13.4% for estradiol. The
interassay variation is quite high, however, the differences between subjects are minimized using ANOVA in
determining the within-individual changes in hormone
levels with time. The sensitivity limit of the assay was 15
pg/ml for progesterone and 1.6 pg/ml for estradiol. All
samples from a given individual were run in the same
assay to minimize the effects of interassay variability.
Participants were divided into three categories according to the frequency of use of betel nut: low (<3 quids/
day), medium (3 quids/day), and high (>3 quid/days).
American Journal of Physical Anthropology—DOI 10.1002/ajpa
TABLE 1. Sample characteristics regarding age, number of years
and frequency of betel nut use, and ingredients included in the
betel quid chewed for the study
Age (X 6 SE)
Number of years chewing betel nut (X 6 SE)
32 6 1.4
11.5 6 2.1
Betel quid chewing frequency
Low (<3 quids/day)
Medium (3 quids/day)
High (>3 quids/day)
Tobacco use in betel quid
Lime use in betel quid
Fig. 2. Average salivary estradiol levels (pg/ml) for samples
collected immediately before and after betel quid use and then
30, 60, 120, 240 min later (n ¼ 11).
TABLE 2. Univariate repeated GLM analyses
Fig. 1. Average salivary progesterone levels (pg/ml) for samples collected immediately before and after betel quid use and
then 30, 60, 120, 240 min later (n ¼ 10).
An univariate repeated measures analysis (GLM with
Huynh-Feldt correction) was used to evaluate whether
there was a short-term effect of chewing betel nut on the
measurement of salivary progesterone and estradiol.
Sample number was entered as a within-subject factor to
assess variation relative to individual baseline levels
while chewing frequency and the presence of two ingredients (tobacco and lime) were entered as between-subject
factors. Age was entered as a covariate. Given the small
final sample size, an a posteriori power analysis was performed using G*Power (Erdfelder et al., 1996) to assess
the possibility of false negatives. Statistical analyses
were performed using SPSS vs. 10.0 for the Macintosh.
The significance level was set at P < 0.05.
Table 1 shows the average age, chewing frequency, and
use of betel nut ingredients by women in the sample.
Of the original 20 participants, only those with hormone levels above the limits of assay detection were
included in the analyses (n ¼ 10 and n ¼ 11 for progesterone and estradiol, respectively)–most of those excluded
were because they were in the follicular rather than
n ¼ 10
n ¼ 11
F ¼ 2.07, df ¼ 2.3,
P ¼ 0.1
F ¼ 1.07, df ¼ 1.8,
P ¼ 0.3
F ¼ 0.08, df ¼ 1,
P ¼ 0.8
F ¼ 0.75, df ¼ 2,
P ¼ 0.5
F ¼ 1.21, df ¼ 1,
P ¼ 0.3
F ¼ 2.07, df ¼ 1,
P ¼ 0.2
F ¼ 0.12, df ¼ 1,
P ¼ 0.7
F ¼ 2.22, df ¼ 2,
P ¼ 0.2
F ¼ 1.17, df ¼ 1,
P ¼ 0.3
F ¼ 0.25, df ¼ 1,
P ¼ 0.6
luteal phase when sampled despite efforts to target only
the luteal phase. Average steroid levels for each sampling
interval are summarized in Figures 1 and 2.
Results for within-subject variation were not significant
for either progesterone or estradiol. Steroid variation
over time relative to baseline levels (sample 1) was not
significant even after age, chewing frequency, tobacco,
and lime use were added as between-subjects factors.
None of the interactions were statistically significant
(Table 2).
The coefficient of variability (SD/mean) of progesterone
measurements immediately after chewing was 98%,
whereas those at 30, 60, 120, and 240 min were 61, 72,
75, and 66%, respectively. Estradiol values were 72% immediately after chewing and 45, 69, 72, and 59% for those
at 30, 60, 120, and 240 min, respectively. For each hormone, paired t-tests revealed that relative to baseline levels, the variation immediately after chewing the betel
quid was not significantly different from that observed at
30, 60, or 120 min.
Observation of the data reveals that, although not statistically significant, the saliva sample given immediately
American Journal of Physical Anthropology—DOI 10.1002/ajpa
after chewing betel quid generally has either elevated or
depressed levels of both hormones relative to the sample
given before chewing indicated by the large 95% confidence limits at this time point. The pattern of variation,
however, is not consistent within groups and varies with
the type of betel quid that was chewed. The high interassay variability may reflect variation in other paan ingredients, time spent chewing, and/or the frequency of use
patterns, but the small sample sizes limits effective analyses of all these variables. Observed differences between
subjects were minimized, however, by using ANOVA to
determine the within-individual changes in hormone levels with time.
Statistical analyses confirm that although post-chewing
steroid levels vary, the variation across time is not significant, even after adjusting for frequency of use and inclusion of tobacco and lime. The cause of the apparent
alteration of steroid levels in saliva immediately after
chewing betel quid is unknown, but it is possible that
some ingredient in the quid interferes with the antibody
reaction of the radioimmunoassay. Variation in steroid
levels following betel quid chewing could also result from
a dilution effect of an increased salivary flow elicited by
ingredients in the quid. Betel-chewers have been shown
to salivate more than non-chewers upon chemical stimulation, with a flow rate positively correlated with the duration of chewing, although no difference in salivary pH
has been observed (Reddy et al., 1971). This suggests that
experienced chewers could secrete more saliva leading to
a concomitant decrease in enzyme, electrolyte and steroid
concentration and contributing to the variation in measured steroids observed among subjects.
The power analysis indicates that the small final sample size in the present study is insufficient to rule out the
possibility of an acute effect of betel nut chewing on salivary steroid levels. However, with this limitation in mind,
our results suggest that even if this were to be the case,
the effect of betel quid chewing on salivary steroid secretions would, as hypothesized, appear to be transitory.
Given the highs and lows in hormone values that immediately follow chewing betel quid we recommend waiting at
least 1 h post quid chewing before collecting saliva samples for measuring these steroids using the techniques
outlined for radioimmunoassay.
Equivalent results to those of the present pilot, were
obtained by Vitzthum et al., (1993) in a small (n ¼ 12) but
comparable study conducted to assess the effect of chewing coca leaves on salivary progesterone secretion among
Bolivian women of reproductive age. Their collection protocol in this study was similar, involving serial saliva
samples at intervals of 15 min, from immediately after
starting chewing coca up until 150 min later. The first
two samples in the series, however, were collected when
coca leaves were still in the mouth. No details on correction for the specific phase of the menstrual cycle at the
time of sample collection were provided.
The Bolivian study also found an apparent transitory
effect from chewing coca leaves, with the highest progesterone levels within the first 30 min following coca leaf
chewing and a return to basal levels immediately after
coca leaves were removed. Variance analysis with repeated
measures found no significant differences between sequential samples except at the 15 min time point, when frequent users showed higher progesterone levels compared
to moderate and rare chewers. The lower interassay variability (12.6%) reported in their study might relate to the
use of a uniform substance–coca leaves, whereas their
intraassay variability (13.8%) is comparable to that reported
here. No information was given with respect to sample
size and statistical power, but the authors recommended
similar modifications to saliva sampling collection protocols to those suggested here in order to account for the
transient variation in salivary hormone values.
This study reveals that betel nut chewing has a transitory effect on salivary progesterone and estradiol levels
analyzed by radioimmunoassay, with values obtained
prior to and then 60 min following betel quid chewing
indistinguishable from each other. These results suggest
that, with specific collection protocols that take into
account time since chewing, it is possible to undertake
salivary steroid analyses of progesterone and estradiol in
populations among which the practice of chewing betel
nut is endemic.
We are grateful to all the women who participated in
the study. We thank the Chadswell Healthy Living
Centre, Hopscotch Asian Women’s Centre, and the Argyll
School for helping with recruitment. We are especially
grateful to Shahreen Ahmed, Sofina Razzaque, Guli
Sarkar, and Kawser Zannath for their enthusiastic
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measurements, progesterone, effect, estradiol, salivary, chewing, nut, betel
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