Betel quid use is associated with anemia among both men and women in Matlab, Bangladesh

Anemia accounts for 8.8% of total disability burden worldwide. Betel quid use among pregnant women has been found to increase anemia risk. Betel quid is prepared by wrapping the betel (or areca) nut, with spices and other additions, in betel or tobacco leaf and it is chewed or placed in the mouth. We explored the association between betel quid use and anemia among men and non-pregnant women. We collected data from a random sample of women and their husbands in Matlab, Bangladesh. Participants reported their current betel quid use and individual characteristics. We assessed hemoglobin (a biomarker of anemia) with a hemoglobinometer and soluble transferrin receptor (a biomarker of iron deficiency) and C-reactive protein (a biomarker of inflammation) in dried blood spots via enzyme immunoassay. We estimated logistic regression models to evaluate the association between betel quid use and anemia and structural equation models (SEM) to evaluate mediating roles of iron deficiency and elevated inflammation. A total of 1133 participants (390 men and 743 non-pregnant women) were included. After controlling for important confounders, any betel quid use was positively associated with anemia among men (OR: 1.80; 95% CI: 1.12, 2.89). Among women, betel quid use was associated with anemia only among the most frequent users (OR: 1.62; 95% CI: 1.03, 2.53). SEM did not reveal indirect paths through inflammation or iron deficiency. Betel quid use may contribute to the burden of anemia among adults in Bangladesh. Our findings suggest the burden of disease attributed to betel quid use has been underestimated.


Introduction
Betel (or areca) nut is the seed of the areca palm (areca catechu) and an addictive stimulant used by 10% of the world's population, primarily in South and Southeast Asia, East Africa, and the Pacific [1,2]. Betel preparations vary by region, but generally involve a quid containing a betel nut wrapped in either a betel or tobacco leaf, which is then placed in the mouth or chewed dry or fresh [3,4]. Reported motivations for betel quid use include improving the smell of the breath, reducing nausea, removing or preventing parasites, satisfying hunger, promoting digestion, or mitigating symptoms of poor mental health [5][6][7].
Betel quid consumption has extensive physiological effects across multiple organ systems, including the nervous, cardiovascular, gastrointestinal, respiratory, endocrine, and reproductive systems [1]. It is associated with preterm birth and low birth weight when used during pregnancy [1,8]. Studies suggest betel quid use is associated with esophageal, liver, and oral cancers, diabetes, hypertension, and cardiovascular disease [1]. With the exception of esophageal and liver cancers, it is not clear that this effect is attributable to betel nut itself, or to other ingredients, such as tobacco, included in many betel quid preparations [9,10]. While research has found an association between betel quid use and both anemia and folate deficiency among pregnant women [11][12][13], whether betel quid use is associated with anemia among the broader population remains an open question.
The global prevalence of anemia is 32.9% and anemia accounts for 8.8% of the total worldwide disability burden [14]. The prevalence of anemia in Bangladesh is substantially higher [15][16][17]. Anemia contributes to declines in physical and cognitive functioning in elderly populations, and reduces productivity among working age adults [14]. Anemia among the working age adult population contributes to reduced labor productivity, continuing cycles of poverty [18].
Betel quid use may cause anemia through multiple pathways, including blood loss, inflammation, and iron deficiency. Although animal studies suggest areca extract may have antiinflammatory effects [19], oral use and the quid preparation likely cause tissue damage and trauma in the mouth and gastrointestinal tract, which may result in inflammation and/or blood loss [20][21][22][23][24], both of which can contribute to anemia [22]. Further, if betel quid use modulates immunity in a way that increases risk for infection, this may contribute to anemia [23,24]. Betel quid's appetite-suppressing effects [25,26] may also reduce food intake, potentially causing anemia via deficiency in iron, folate, and other micronutrients [13].
Public health experts have called for increased research on the health effects of betel quid use [27]. Here, we contribute to this body of research by investigating associations between betel quid use and anemia among men and non-pregnant women, controlling for important socioeconomic confounders. In addition, we assessed two potential causal pathways, inflammation and iron deficiency.

Research setting
Data were collected in rural Matlab, Bangladesh. The region is undergoing rapid economic development and an epidemiological transition in which infectious disease rates have fallen while chronic diseases have become more prevalent, and rates of both undernutrition and obesity are high [28][29][30]. Betel quid is commonly used by both men and women in Matlab. While betel quid is widely consumed in small quantities at festive events, daily use of larger quantities is perceived in our study population (and documented among our study participants) to be more common among those with less education (p<0.001) and in older age groups (p<0.001).

Study design and participants
Participants were originally recruited in 2010 by Shenk and Alam, when 944 women were randomly selected from a population roster housed by icddr,b (formerly known as the also categorized by frequency of reported use: not a current user, infrequent user (at least once monthly but less than daily), low daily user (less than five times per day), or high daily user (at least five times per day), to examine effects of heavy use and the dose-response nature of any associations.
Adjustment variables were age, education, MacArthur Ladder, food security, food source, tobacco use, and exposure to secondhand tobacco smoke. We used both MacArthur Ladder and education to adjust for socioeconomic status [45,46]. We avoided use of current income, because it could substantially underestimate the wealth of older participants who relied primarily on support from adult children, rather than their own income. We also examined household asset score as a control variable instead of MacArthur Ladder, which did not change our findings and did not improve the model fit.
We used logistic regression to estimate crude and adjusted odds ratios. We also estimated logistic regression models with potential mediator variables (elevated inflammation and iron deficiency). Finally, we used structural equation modeling (SEM) to estimate path coefficients to describe the effects of mediator variables: elevated inflammation and iron deficiency. All data were analyzed in SAS 9.4.

Research ethics approval
The study was approved by the Ethical Review Committee at icddr,b and the Pennsylvania State University Institutional Review Board. Participants were informed the study was voluntary and provided written consent to participate; they were also provided with their anemia status, height, weight, BMI, and other health information at the time of data collection.

Results
Anemia data were available for 1133 participants (390 men and 743 women; Table 1). A greater proportion of women were anemic (48.6%) compared with men (40.3%). Betel quid was used by 51.8% of men and 53.3% of women; more men (33.1%) than women (21.2%) reported the highest category of betel quid use (at least five times a day) (p<0.001). Men were older (p<0.001), had more years of education (p = 0.009), smoked tobacco at higher rates (p<0.001), and were more likely to be underweight (p<0.001) than women. Women reported higher scores on MacArthur Ladder (p<0.001), more often obtained all of their food from the bazaar (p<0.001), and were exposed to passive smoke at higher rates (p<0.001) than men.
Gender interacted with betel quid use in logistic regression models of anemia after controlling for confounders common for both men and women (coefficient for betel quid use*gender: 0.634; p: 0.019), therefore subsequent modeling was stratified by gender. We assessed interactions between betel quid use and age for men and women and did not identify any.
Underweight was independently positively associated with anemia among women (OR: 1.77; 95% CI: 1.05, 3.00), but not men (OR: 1.47; 95% CI: 0.81, 2.66), after controlling for confounders and mediators (S1 Table). However, because underweight is not itself a cause of anemia but instead a correlate of iron and other micronutrient deficiencies, the interpretability of these models is limited.

Discussion
Betel quid use was associated with anemia among men and non-pregnant women, although the association in women was limited to the most frequent users. Tests for trend suggest a dose-response relationship (increasing frequency of betel quid use was associated with increased risk for anemia) among both men and women. These observations suggest betel quid use may increase anemia risk for both genders. When we investigated direct and indirect PLOS GLOBAL PUBLIC HEALTH pathways, we found evidence for both direct effects, and, among women, a pathway through iron deficiency. Our finding that betel quid use may contribute to anemia among men and non-pregnant women adds needed breadth to the current research regarding health effects of betel quid use, which has largely focused on betel quid's association with adverse pregnancy outcomes as well as oropharyngeal and gastrointestinal cancers. Increasing risk for anemia may be one of betel quid's most impactful effects on users' health, as anemia reduces the ability to work and contributes to risk for other illnesses. The high prevalence of anemia in Bangladesh, and our findings showing direct and indirect pathways from betel quid use to anemia, suggest the disease burden attributable to betel quid use may be substantially higher than previously appreciated.
Multiple physiological pathways might explain the direct effects of betel quid use and anemia suggested by our findings. Betel quid use might cause irritation or blood loss in the mouth or gastrointestinal tract, which could lead to anemia [47,48]. Using betel quid might also have direct physiological effects on hemoglobin production, erythropoiesis, or erythrocyte longevity, or adversely affect these via oxidative stress [49]. Betel quid use might also act through mediating variables not considered here, such as immune suppression and increased risk for infections that can cause anemia [50]. In addition, tannins or other components of betel quid may cause damage to tissues of the gastrointestinal tract leading to blood loss.

PLOS GLOBAL PUBLIC HEALTH
There are several important strengths to this analysis, including its large sample size, the evaluation of direct and indirect pathways through inflammation and iron deficiency, and the inclusion of important covariates. However, we note several important limitations. Our ability to make causal inference is limited by the cross-sectional (we cannot be certain betel quid use preceded anemia) and observational (participants were not randomized to receive betel quid) nature of the study design. Some of the associations we observed had wide confidence intervals and/or confidence intervals that included (or came close to including) the null effect; this may be due to small numbers (e.g., of women in the highest frequency category of betel quid use) and should be interpreted with caution. The survey data relied on self-report and may be vulnerable to bias (some participants may have been more likely than others to under-report betel quid use). Additionally, our analyses are limited by the potential for unmeasured confounding due to infectious diseases (e.g., hookworm), other components of the diet not collected through the survey, and contraceptive use. This study did not capture how participants typically prepared betel quid, therefore we cannot speak to how individual ingredients might contribute to anemia-it is possible a non-betel ingredient, like chewing tobacco, explains some or all of the association between betel quid use and anemia [13,51,52]. Furthermore, there may be pathways through inflammation or appetite suppression that we were not able to capture with biomarkers of inflammation and iron deficiency. It is also important to note the measurement of inflammation used in this study, CRP, is imperfect, and is not the only available biomarker of inflammation. Therefore, our data may have partially misclassified inflammation within our study population. The biomarker of iron deficiency we used, sTfR, while ideal in many ways for population-based research in remote settings, lacks consensus around a single definition of iron deficiency [53] and so may have misclassified some cases, potentially obscuring a pathway through iron deficiency. However, this kind of misclassification is expected to PLOS GLOBAL PUBLIC HEALTH be non-differential, biasing results toward the null hypothesis, yet despite this we were clearly able to detect an association. Finally, the generalizability of our findings beyond rural Bangladesh remains to be evaluated.

Conclusions
Our findings suggest betel quid use may increase risk for anemia. Current research underestimates the disease burden attributable to betel quid use particularly for men and non-pregnant women, among whom the burden of anemia remains substantial but chronically understudied, and for whom impairment or disability due to anemia is likely to have negative health and economic consequences for individuals and their families.   PLOS GLOBAL PUBLIC HEALTH Supporting information S1 Table. Logistic regression models of anemia including underweight. (DOCX)