Calcium-containing Eggshell Powder Supplementation to Mitigate Toxic Effects of Excess Fluoride Intake among Women in Ethiopian Rift Valley: Efficacy, association, safety and acceptability

Abstract

Background: Worldwide, 50 million people suffer from fluorosis, which affects not only teeth, but also bones, joints, gut and brain functions. In Ethiopia, where defluoridation requires costly infrastructure, more than 14 million individuals, mainly in the Rift Valley, are affected by fluorosis. Drinking water sources, in the Rift Valley of Ethiopia, contain fluoride (F) levels exceeding the World Health Organization (WHO) limit of 1.5mg/L. F exposure may be an added concern for women’s bone and dental health where there is low Ca intake. Studies suggest that the adverse effects of F can be reversed or lessened by providing sufficient food intake of protein, calcium (Ca), anti-oxidants and vitamin D. Of these, Ca is the most studied by ecological studies. However, there have been no intervention studies of Ca to mitigate fluorosis at the community level in Ethiopia. We therefore hypothesized that supplementation of an age old, sustainable and low cost source of Ca, i.e., eggshell, as a dietary Ca source would mitigate the toxic effects of excess F intake in women. Thus, the aim of this study was to test the efficacy of calcium-containing ESP supplementation, as a proof of concept, to reduce F absorption as measured by urinary F excretion (a primary outcome measure) and mitigation of fluorosis symptoms (secondary outcomes) in women living in a fluorosis-affected area. The overall objective of this PhD research was to assess efficacy, association and safety and acceptability of calcium-containing eggshell powder supplementation to mitigate toxic effects of excess fluoride intake among women in Ethiopian Rift Valley. Methods: Women (n=270) from two villages provided clinical and questionnaire data for the cross-sectional survey. Dental fluorosis examination was done using Dean’s Index, and skeletal and non-skeletal fluorosis assessment was carried out using physical tests and clinical symptoms. Daily Ca intake was estimated by a food frequency questionnaire. Food, drinking water and beverage samples were analyzed for F level. Eighty-two women (41 Intervention Group, IG; 41 Control Group, CG) were recruited for the Phase II clinical trial; 39 in each group completed the trial. Morning spot urine was collected for testing fluoride (F), calcium (Ca) and creatinine concentrations, before (baseline, BL) and after (endline, EL) the intervention that was 6-months daily supplementation with 2.4 gram calcium-containing eggshell powder/ESP (providing ~1000 mg of calcium). Dental, skeletal and non-skeletal fluorosis assessments were carried out at BL and, except for dental, at EL. For the purpose of safety and acceptability assessment, body retention of iron and zinc were measured using blood levels, prior to and after the calcium containing ESP supplementation among the study subjects. Occurrence of side effects such as nausea, vomiting, constipation, and abdominal bloating and gas related to excess calcium intake in the Phase II trial were assessed using checklists on a monthly period. In addition, blood tests for malaria, hemoglobin and hematocrit were done immediately in the field. Descriptive statistics, bivariate and multivariable logistic regression, relative risk (RR), paired samples t-test and two independent samples t-test, linear generalized estimating equation (GEE) and multivariate analysis of the GEE model were used to analyze and compare outcomes between groups. Results: Many subjects (56.3%) exhibited dental fluorosis. Women having ≤ 400 mg/day Ca intake had ~3 times greater odds of developing skeletal rigidity with joint pains [AOR=2.8, 95%CI: 1.6, 5.0] and muscular weakness [AOR=2.9, 95%CI: 1.3, 6.3] compared to those with higher intakes. At EL, women in the IG had about six-fold lower urinary F excretion [β = - 6.1 (95% CI: -7.1, -5.1)] compared to women in the CG. The risk of developing skeletal fluorosis tested using the ability to bend body and touch floor or toe [RR = 0.21 (95% CI: 0.07, 0.69)], and stretch and fold arms to touch back of head [RR = 0.18 (95% CI: 0.04, 0.77)] were significantly reduced in the intervention group by 79% and 82% respectively compared with the control. Majority of the women in IG reported mitigation of pain and muscular symptoms of non-skeletal fluorosis ranging from lowest RR = 0.17 (95% CI: 0.05, 0.52) to highest RR = 0.59 (95% CI: 0.39, 0.88) after the calcium-containing ESP supplementation than in CG. Conclusion: Signs and symptoms of dental, skeletal and non-skeletal fluorosis were prevalent in women of child-bearing age in this area of the Rift Valley of Ethiopia. As low dietary Ca intake was significantly associated with symptoms related to skeletal and non-skeletal fluorosis, this warrants nutritional intervention on calcium intakes in this setting. A significant reduction in urinary F excretion and reduction in many fluorosis symptoms were observed among women supplemented with calcium-containing ESP, thus providing evidence for using this dietary calcium (ESP) source for mitigation of fluorosis. Safety and acceptability assessments conducted as part of the study indicated that calcium-containing eggshell powder supplementation was generally accepted, well-tolerated and did not pose significant risks to the health of participants. The potential feasibility, sustainability and safety of using home prepared crushed eggshells in areas where fluorosis is endemic need to be studied. Clinical trials registration: NCT03355222 Keywords: Calcium; Fluoride; Fluorosis; Eggshell powder; Safety; Acceptibility; Women; Ethiopia; Rift Valley.