Knowledge gaps in understanding the metabolic and clinical effects of excess folates/folic acid: a summary, and perspectives, from an NIH workshop.

Folate, an essential nutrient found naturally in foods in a reduced form, is present in dietary supplements and fortified foods in an oxidized synthetic form (folic acid). There is widespread agreement that maintaining adequate folate status is critical to prevent diseases due to folate inadequacy (e.g., anemia, birth defects, and cancer). However, there are concerns of potential adverse effects of excess folic acid intake and/or elevated folate status, with the original concern focused on exacerbation of clinical effects of vitamin B-12 deficiency and its role in neurocognitive health. More recently, animal and observational studies have suggested potential adverse effects on cancer risk, birth outcomes, and other diseases. Observations indicating adverse effects from excess folic acid intake, elevated folate status, and unmetabolized folic acid (UMFA) remain inconclusive; the data do not provide the evidence needed to affect public health recommendations. Moreover, strong biological and mechanistic premises connecting elevated folic acid intake, UMFA, and/or high folate status to adverse health outcomes are lacking. However, the body of evidence on potential adverse health outcomes indicates the need for comprehensive research to clarify these issues and bridge knowledge gaps. Three key research questions encompass the additional research needed to establish whether high folic acid or total folate intake contributes to disease risk. 1) Does UMFA affect biological pathways leading to adverse health effects? 2) Does elevated folate status resulting from any form of folate intake affect vitamin B-12 function and its roles in sustaining health? 3) Does elevated folate intake, regardless of form, affect biological pathways leading to adverse health effects other than those linked to vitamin B-12 function? This article summarizes the proceedings of an August 2019 NIH expert workshop focused on addressing these research areas.

Children with Poor Linear Growth Are at Risk for Repeated Relapse to Wasting after Recovery from Moderate Acute Malnutrition.

Background: Nutrition programs frequently approach wasting and stunting as 2 separate conditions with distinct causes and effects. Although several cross-sectional studies have identified an association between the 2 conditions, longitudinal studies are useful to quantify the risk of acute malnutrition based on the trajectory of linear growth. Objective: We analyzed data from a longitudinal study to explore associations between linear growth and relapse to acute malnutrition in high-risk children during the year after recovery from moderate acute malnutrition (MAM). Methods: This was a secondary data analysis from a cluster randomized trial involving 1487 Malawian children 6-62 mo old treated for MAM and enrolled upon recovery. Children were followed for 1 y, during which data were collected on anthropometric progress, symptoms of illness, and household food security. Multivariate fixed-effects logistic regression was used to identify associations between linear growth and relapse to acute malnutrition. Results: Children who have recovered from MAM proved to be a high-risk population, with nearly half experiencing a decrease in height-for-age z score (HAZ) for 12 mo. Children whose HAZ was declining were more likely to relapse to MAM or SAM than were those whose linear growth rate maintained or increased their HAZ (P < 0.001). Mean changes of +0.15, -0.03, -0.17, and -0.53 in HAZ were observed for those who sustained recovery, relapsed to MAM once, relapsed to MAM multiple times, and developed SAM, respectively. Conclusion: Our results add to the body of evidence suggesting that acute wasting is a harbinger of subsequent stunting. Children who experience poor linear growth after MAM are more likely to experience relapse. Given this bidirectional relation between wasting and stunting, supplementary feeding programs should consider both when designing protocols, aiming to optimize linear growth and achieve acute weight gain, as a means of reducing relapse. This trial was registered at clinicaltrials.gov as NCT02351687.

Choline and its metabolites are differently associated with cardiometabolic risk factors, history of cardiovascular disease, and MRI-documented cerebrovascular disease in older adults.

Background: There is a potential role of choline in cardiovascular and cerebrovascular disease through its involvement in lipid and one-carbon metabolism.Objective: We evaluated the associations of plasma choline and choline-related compounds with cardiometabolic risk factors, history of cardiovascular disease, and cerebrovascular pathology.Design: A cross-sectional subset of the Nutrition, Aging, and Memory in Elders cohort who had undergone MRI of the brain (n = 296; mean ± SD age: 73 ± 8.1 y) was assessed. Plasma concentrations of free choline, betaine, and phosphatidylcholine were measured with the use of liquid-chromatography-stable-isotope dilution-multiple-reaction monitoring-mass spectrometry. A volumetric analysis of MRI was used to determine the cerebrovascular pathology (white-matter hyperintensities and small- and large-vessel infarcts). Multiple linear and logistic regression models were used to examine relations of plasma measures with cardiometabolic risk factors, history of cardiovascular disease, and radiologic evidence of cerebrovascular pathology.Results: Higher concentrations of plasma choline were associated with an unfavorable cardiometabolic risk-factor profile [lower high-density lipoprotein (HDL) cholesterol, higher total homocysteine, and higher body mass index (BMI)] and greater odds of large-vessel cerebral vascular disease or history of cardiovascular disease but lower odds of small-vessel cerebral vascular disease. Conversely, higher concentrations of plasma betaine were associated with a favorable cardiometabolic risk-factor profile [lower low-density lipoprotein (LDL) cholesterol and triglycerides] and lower odds of diabetes. Higher concentrations of plasma phosphatidylcholine were associated with characteristics of both a favorable cardiometabolic risk-factor profile (higher HDL cholesterol, lower BMI, lower C-reactive protein, lower waist circumference, and lower odds of hypertension and diabetes) and an unfavorable profile (higher LDL cholesterol and triglycerides).Conclusion: Choline and its metabolites have differential associations with cardiometabolic risk factors and subtypes of vascular disease, thereby suggesting differing roles in the pathogenesis of cardiovascular and cerebral large-vessel disease compared with that of small-vessel disease.

Excessive folic acid intake and relation to adverse health outcome.

The recent increase in the intake of folic acid by the general public through fortified foods and supplements, has raised safety concern based on early reports of adverse health outcome in elderly with low B12 status who took high doses of folic acid. These safety concerns are contrary to the 2015 WHO statement that "high folic acid intake has not reliably been shown to be associated with negative healeffects". In the folic acid post-fortification era, we have shown that in elderly participants in NHANES 1999-2002, high plasma folate level is associated with exacerbation of both clinical (anemia and cognitive impairment) and biochemical (high MMA and high Hcy plasma levels) signs of vitamin B12 deficiency. Adverse clinical outcomes in association with high folate intake were also seen among elderly with low plasma B12 levels from the Framingham Original Cohort and in a study from Australia which combined three elderly cohorts. Relation between high folate and adverse biochemical outcomes were also seen in the Sacramento Area Latino Study on Aging (High Hcy, high MMA and lower TC2) and at an outpatient clinic at Yale University where high folate is associated with higher MMA in the elderly but not in the young. Potential detrimental effects of high folic acid intake may not be limited to the elderly nor to those with B12 deficiency. A study from India linked maternal high RBC folate to increased insulin resistance in offspring. Our study suggested that excessive folic acid intake is associated with lower natural killer cells activity in elderly women. In a recent study we found that the risk for unilateral retinoblastoma in offspring is 4 fold higher in women that are homozygotes for the 19 bp deletion in the DHFR gene and took folic acid supplement during pregnancy. In the elderly this polymorphism is associated with lower memory and executive scores, both being significantly worse in those with high plasma folate. These and other data strongly imply that excessive intake of folic acid is not always safe in certain populations of different age and ethnical/genetic background.

B vitamins and the aging brain.

Deficiencies of the vitamins folate, B(12) , and B(6) are associated with neurological and psychological dysfunction and with congenital defects. In the elderly, cognitive impairment and incident dementia may be related to the high prevalence of inadequate B vitamin status and to elevations of plasma homocysteine. Plausible mechanisms include homocysteine neurotoxicity, vasotoxicity, and impaired S-adenosylmethionine-dependent methylation reactions vital to central nervous system function. In light of this, it is imperative to find safe ways of improving vitamin B status in the elderly without exposing some individuals to undue risk.

Circulating unmetabolized folic acid and 5-methyltetrahydrofolate in relation to anemia, macrocytosis, and cognitive test performance in American seniors.

BACKGROUND: Folate deficiency has serious consequences for the fetus. Folic acid fortification of food addresses this problem. However, clinical consequences of vitamin B-12 deficiency may be worsened by high folic acid intakes, perhaps as a direct result of unmetabolized folic acid, which does not occur naturally in body tissues. OBJECTIVE: We attempted to attribute associations that we previously found between higher folate status and anemia and cognitive test performance to circulating unmetabolized folic acid or 5-methyltetrahydrofolate (5MeTHF). DESIGN: The subjects (n = 1858) were senior participants in the US National Health and Nutrition Examination Survey (1999-2002) who had normal renal function and reported no history of stroke, recent anemia therapy, or diseases of the liver, thyroid, or coronary arteries. Subjects had undergone a phlebotomy, a complete blood count, and cognitive and dietary assessments. RESULTS: Circulating unmetabolized folic acid was detected in approximately 33% of the subjects and was related to an increased odds of anemia in alcohol users. In seniors with a serum vitamin B-12 concentration <148 pmol/L or a plasma methylmalonic acid concentration > or =210 nmol/L, the presence compared with the absence of detectable circulating unmetabolized folic acid was related to lower cognitive test scores and lower mean cell volume. In the same subgroup, higher serum 5MeTHF was related to an increased odds of anemia and a marginally significantly decreased odds of macrocytosis. In seniors with a normal vitamin B-12 status, a higher serum 5MeTHF concentration was related to higher cognitive test scores. CONCLUSION: Results of this epidemiologic study were somewhat consistent with reports on the folic acid treatment of patients with pernicious anemia, but some findings were unexpected.

Community-level micronutrient fortification of school lunch meals improved vitamin A, folate, and iron status of schoolchildren in Himalayan villages of India.

Anemia and micronutrient deficiencies are common among Indian schoolchildren. We assessed the effectiveness of micronutrient fortification of meals cooked and fortified at school on anemia and micronutrient status of schoolchildren in Himalayan villages of India. In this placebo-controlled, cluster-randomized study, 499 schoolchildren (6-10 y) received either multiple micronutrients (treatment group) or placebo (control group) as part of school meals (6 d/wk) for 8 mo. Both groups were dewormed at the beginning of the study. The micronutrient premix provided 10 mg iron, 375 microg vitamin A, 4.2 mg zinc, 225 microg folic acid, and 1.35 microg vitamin B-12 for each child per day (approximately 75% recommended dietary allowance). Blood samples drawn before and after the intervention were analyzed for hemoglobin, ferritin, retinol, zinc, folate, and vitamin B-12. Baseline prevalence of anemia (37%), iron deficiency anemia (10%), low serum ferritin (24%), retinol (56%), zinc (74%), folate (68%), and vitamin B-12 (17%) did not differ between groups. Postintervention, fewer in the treatment group had lower serum retinol [odds ratio (OR) (95% CI): 0.57 (0.33-0.97)] and folate [OR (95% CI): 0.47 (0.26-0.84)] than the control group. The serum vitamin B-12 concentration decreased in both groups, but the magnitude of change was less in the treatment than in the control group (P < 0.05). Total body iron (TBI) increased in both groups; however, the change was greater in the treatment than in the control group (P < 0.05). Micronutrient fortification of school meals by trained school personnel was effective in improving vitamin A, folate, and TBI status while also reducing the magnitude of a decrease in vitamin B-12 status.

MAT1A variants are associated with hypertension, stroke, and markers of DNA damage and are modulated by plasma vitamin B-6 and folate.

BACKGROUND: The S-adenosylmethionine synthetase type 1 (MAT1A) gene encodes a key enzyme in one-carbon nutrient metabolism. OBJECTIVE: This study aimed to determine the association of MAT1A variants with homocysteine, DNA damage, and cardiovascular disease (CVD). DESIGN: Eight variants of MAT1A were examined for associations with hypertension, stroke, CVD, homocysteine, and DNA damage in 1006 participants of the Boston Puerto Rican Health Study. Two variants were replicated in 1147 participants of the Nutrition, Aging, and Memory in Elders Study. RESULTS: Two variants and haplotypes were strongly associated with hypertension and stroke, independent of methylenetetrahydrofolate reductase (MTHFR) variants. Homozygotes of the MAT1A d18777A (rs3851059) allele had a significantly greater likelihood of stroke (odds ratio: 4.30; 95% CI: 1.34, 12.19; P = 0.006), whereas 3U1510A (rs7087728) homozygotes had a lower likelihood of hypertension (odds ratio: 0.67; 95% CI: 0.48, 0.95; P = 0.022) and stroke (odds ratio: 0.35; 95% CI: 0.15, 0.82; P = 0.015). A similar trend of association was observed in a second elderly population. Furthermore, strong interactions between MAT1A genotypes and vitamin B-6 status were found. Carriers of the nonrisk allele 3U1510A had a lower 8-hydroxydeoxyguanosine concentration--a biomarker of oxidative DNA damage--when plasma vitamin B-6 was high, whereas homozygotes for the risk-allele 3U1510G had higher 8-hydroxydeoxyguanosine concentrations, regardless of vitamin B-6 status. CONCLUSIONS: MAT1A variants were strongly associated with hypertension and stroke. Improving folate and vitamin B-6 status might decrease the CVD risk of only a subset of the population, depending on genotype. These findings suggest that impairments in methylation activity, independent of homocysteine, have an effect on CVD risk.

Examination of selected national policies towards mandatory folic acid fortification.

The purpose of this paper is to present an examination of the contrasting policies towards mandatory folic acid fortification in six countries from different regions of the world. Three questions are addressed: 1) What is the policy of the country? 2) Why was the policy adopted? 3) What lessons have been learned? Policy contrasts among countries were assessed as reflecting different interpretations of the potential risks and benefits associated with folic acid fortification. Although commonalities were identified, it was considered unlikely that there could be a standard policy response for all countries. Instead, a country-by-country policy response based on national circumstances is indicated.

Not all cases of neural-tube defect can be prevented by increasing the intake of folic acid.

Some countries have introduced mandatory folic acid fortification, whereas others support periconceptional supplementation of women in childbearing age. Several European countries are considering whether to adopt a fortification policy. Projections of the possible beneficial effects of increased folic acid intake assume that the measure will result in a considerable reduction in neural-tube defects (NTD) in the target population. Therefore, the objective of the present study is to evaluate the beneficial effects of different levels of folic acid administration on the prevalence of NTD. Countries with mandatory fortification achieved a significant increase in folate intake and a significant decline in the prevalence of NTD. This was also true for supplementation trials. However, the prevalence of NTD at birth declined to approximately five cases at birth per 10 000 births and seven to eight cases at birth or abortion per 10 000 births. This decline was independent of the amount of folic acid administered and apparently reveals a 'floor effect' for folic acid-preventable NTD. This clearly shows that not all cases of NTD are preventable by increasing the folate intake. The relative decline depends on the initial NTD rate. Countries with NTD prevalence close to the observed floor may have much smaller reductions in NTD rates with folic acid fortification. Additionally, potential adverse effects of fortification on other vulnerable population groups have to be seriously considered. Policy decisions concerning national mandatory fortification programmes must take into account realistically projected benefits as well as the evidence of risks to all vulnerable groups.

Cognitive impairment in folate-deficient rats corresponds to depleted brain phosphatidylcholine and is prevented by dietary methionine without lowering plasma homocysteine.

Poor folate status is associated with cognitive decline and dementia in older adults. Although impaired brain methylation activity and homocysteine toxicity are widely thought to account for this association, how folate deficiency impairs cognition is uncertain. To better define the role of folate deficiency in cognitive dysfunction, we fed rats folate-deficient diets (0 mg FA/kg diet) with or without supplemental L-methionine for 10 wk, followed by cognitive testing and tissue collection for hematological and biochemical analysis. Folate deficiency with normal methionine impaired spatial memory and learning; however, this impairment was prevented when the folate-deficient diet was supplemented with methionine. Under conditions of folate deficiency, brain membrane content of the methylated phospholipid phosphatidylcholine was significantly depleted, which was reversed with supplemental methionine. In contrast, neither elevated plasma homocysteine nor brain S-adenosylmethionine and S-adenosylhomocysteine concentrations predicted cognitive impairment and its prevention by methionine. The correspondence of cognitive outcomes to changes in brain membrane phosphatidylcholine content suggests that altered phosphatidylcholine and possibly choline metabolism might contribute to the manifestation of folate deficiency-related cognitive dysfunction.

Nutrition and Crohn's disease: an update of print and Web-based guidance.

This article reviews the role of nutrition in Crohn's disease, one of the inflammatory bowel diseases. In addition to presenting a comprehensive review of the state-of-the-evidence on nutritional risks and nutritional therapies in Crohn's disease, and making specific nutrient recommendations, this article includes a list of Web-based resources, including websites, blogs, newsletters, and multimedia podcasts that can be utilized by patients and healthcare providers alike to learn more about the etiology, pathophysiology, and nutritional management of Crohn's disease.

A temporal association between folic acid fortification and an increase in colorectal cancer rates may be illuminating important biological principles: a hypothesis.

Nationwide fortification of enriched uncooked cereal grains with folic acid began in the United States and Canada in 1996 and 1997, respectively, and became mandatory in 1998. The rationale was to reduce the number of births complicated by neural tube defects. Concurrently, the United States and Canada experienced abrupt reversals of the downward trend in colorectal cancer (CRC) incidence that the two countries had enjoyed in the preceding decade: absolute rates of CRC began to increase in 1996 (United States) and 1998 (Canada), peaked in 1998 (United States) and 2000 (Canada), and have continued to exceed the pre-1996/1997 trends by 4 to 6 additional cases per 100,000 individuals. In each country, the increase in CRC incidence from the prefortification trend falls significantly outside of the downward linear fit based on nonparametric 95% confidence intervals. The statistically significant increase in rates is also evident when the data for each country are analyzed separately for men and women. Changes in the rate of colorectal endoscopic procedures do not seem to account for this increase in CRC incidence. These observations alone do not prove causality but are consistent with the known effects of folate on existing neoplasms, as shown in both preclinical and clinical studies. We therefore hypothesize that the institution of folic acid fortification may have been wholly or partly responsible for the observed increase in CRC rates in the mid-1990s. Further work is needed to definitively establish the nature of this relationship. In the meantime, deliberations about the institution or enhancement of fortification programs should be undertaken with these considerations in mind.

Folate and vitamin B-12 status in relation to anemia, macrocytosis, and cognitive impairment in older Americans in the age of folic acid fortification.

BACKGROUND: Historic reports on the treatment of pernicious anemia with folic acid suggest that high-level folic acid fortification delays the diagnosis of or exacerbates the effects of vitamin B-12 deficiency, which affects many seniors. This idea is controversial, however, because observational data are few and inconclusive. Furthermore, experimental investigation is unethical. OBJECTIVE: We examined the relations between serum folate and vitamin B-12 status relative to anemia, macrocytosis, and cognitive impairment (ie, Digit Symbol-Coding score < 34) in senior participants in the 1999-2002 US National Health and Nutrition Examination Survey. DESIGN: The subjects had normal serum creatinine concentrations and reported no history of stroke, alcoholism, recent anemia therapy, or diseases of the liver, thyroid, or coronary arteries (n = 1459). We defined low vitamin B-12 status as a serum vitamin B-12 concentration < 148 pmol/L or a serum methylmalonic acid concentration > 210 nmol/L-the maximum of the reference range for serum vitamin B-12-replete participants with normal creatinine. RESULTS: After control for demographic characteristics, cancer, smoking, alcohol intake, serum ferritin, and serum creatinine, low versus normal vitamin B-12 status was associated with anemia [odds ratio (OR): 2.7; 95% CI: 1.7, 4.2], macrocytosis (OR: 1.8; 95% CI: 1.01, 3.3), and cognitive impairment (OR: 2.5; 95% CI: 1.6, 3.8). In the group with a low vitamin B-12 status, serum folate > 59 nmol/L (80th percentile), as opposed to < or = 59 nmol/L, was associated with anemia (OR: 3.1; 95% CI: 1.5, 6.6) and cognitive impairment (OR: 2.6; 95% CI: 1.1, 6.1). In the normal vitamin B-12 group, ORs relating high versus normal serum folate to these outcomes were < 1.0 (P(interaction) < 0.05), but significantly < 1.0 only for cognitive impairment (0.4; 95% CI: 0.2, 0.9). CONCLUSION: In seniors with low vitamin B-12 status, high serum folate was associated with anemia and cognitive impairment. When vitamin B-12 status was normal, however, high serum folate was associated with protection against cognitive impairment.

Breakfast cereal fortified with folic acid, vitamin B-6, and vitamin B-12 increases vitamin concentrations and reduces homocysteine concentrations: a randomized trial.

BACKGROUND: High homocysteine and low B vitamin concentrations have been linked to the risk of vascular disease, stroke, and dementia and are relatively common in older adults. OBJECTIVE: We assessed the effect of breakfast cereal fortified with folic acid, vitamin B-6, and vitamin B-12 on vitamin and homocysteine status. DESIGN: A randomized, double-blind trial was conducted in 189 volunteers aged 50-85 y. The subjects had no history of hypertension, anemia, asthma, cancer, or cardiovascular or digestive disease and did not regularly consume multiple or B vitamin supplements or highly fortified breakfast cereal. Subjects were randomly assigned to consume 1 cup (0.24 L) breakfast cereal fortified with 440 microg folic acid, 1.8 mg vitamin B-6, and 4.8 microg vitamin B-12 or placebo cereal for 12 wk. Blood was drawn at 0, 2, 12, and 14 wk. Methionine-loading tests were conducted at baseline and week 14. RESULTS: Final baseline-adjusted plasma homocysteine concentrations were significantly lower and B vitamin concentrations were significantly higher in the treatment group than in the placebo group (P < 0.001). The percentage of subjects with plasma folate concentrations < 11 nmol/L decreased from 2% to 0%, with vitamin B-12 concentrations < 185 pmol/L from 9% to 3%, with vitamin B-6 concentrations < 20 nmol/L from 6% to 2%, and with homocysteine concentrations > 10.4 micromol/L (women) or > 11.4 micromol/L (men) from 6.4% to 1.6%. The percentage of control subjects with values beyond these cutoff points remained nearly constant or increased. CONCLUSIONS: In this relatively healthy group of volunteers, consumption of 1 cup fortified breakfast cereal daily significantly increased B vitamin and decreased homocysteine concentrations, including post-methionine-load homocysteine concentrations.

Plasma homocysteine, hypertension incidence, and blood pressure tracking: the Framingham Heart Study.

Plasma homocysteine is cross-sectionally associated with blood pressure in large, community-based studies. It is unknown whether elevated plasma homocysteine predicts hypertension incidence. We investigated the relations of baseline plasma total homocysteine levels to hypertension incidence and blood pressure tracking in 2104 Framingham Heart Study participants (mean age, 57 years; 58% women), who were free of hypertension, myocardial infarction, heart failure, atrial fibrillation, or renal failure at baseline. Baseline mean+/-SD plasma homocysteine was 10.1+/-3.7 micromol/L. On follow-up 4 years from baseline, 360 persons (17.1%) had developed hypertension, and 878 persons (41.7%) had progressed to a higher blood pressure stage. In unadjusted analyses, a 1-SD higher log homocysteine value was associated with increased odds of developing hypertension (odds ratio [OR], 1.18; 95% confidence interval [CI], 1.05 to 1.32) and increased odds of blood pressure progression (OR, 1.17; 95% CI, 1.07 to 1.27). The relations of plasma homocysteine to the incidence of hypertension or blood pressure progression were statistically nonsignificant in age- and sex-adjusted logistic regression models (OR, 0.98; 95% CI, 0.87 to 1.11 and OR, 1.05; 95% CI, 0.96 to 1.16, respectively) and in multivariable models adjusted for age, sex, body mass index, diabetes, interim weight change, smoking, serum creatinine, baseline blood pressure, and blood pressure category (OR, 0.92; 95% CI, 0.81 to 1.06 and OR, 1.07; 95% CI, 0.97 to 1.18, respectively). In conclusion, we found no major relation of baseline plasma homocysteine levels to hypertension incidence or longitudinal blood pressure progression in a large, community-based cohort of nonhypertensive individuals after adjustment for age, sex, and other important covariates.

Tropical enteritis: nutritional consequences and connections with the riddle of cholera.

One of the important consequences of the infection-nutrition interaction is mediated by malabsorption associated with chronic inflammation in the intestine, enteritis. Studies made possible after development of the peroral intestinal biopsy technique in the 1950s indicated the wide prevalence of enteropathy, particularly in tropical developing countries with poor levels of sanitation. Some consider this so-called subclinical tropical malabsorption to be the base of an iceberg, whose tip is tropical sprue, a severe form of malabsorption leading to nutritional deficiency that had been reported in colonial expatriates in tropical countries for 200 y. Some of the first demonstrations of the prevalence of tropical enteritis in Asia were made in quest of the pathologic lesion of cholera, and further examination of the water and electrolyte, as well as nutrient, malabsorption in cholera led serendipitously to the discovery of the oral rehydration solution for the treatment of diarrheal disease.