author: Christopher Gardner; Judith Wylie-Rosett; Samuel S. Gidding; Lyn M. Steffen; Rachel K. Johnson; Diane Reader; Alice H. Lichtenstein; [^1][^2][^3][^4][^5]
date: 2012-08
references:
subtitle: : A Scientific Statement from the American Heart Association and the American Diabetes Association
title: Nonnutritive Sweeteners: Current Use and Health Perspectives

A 2009 American Heart Association scientific statement titled "Dietary Sugars Intake and Cardiovascular Health" (1) concluded that current intake of added sugars among Americans greatly exceeds discretionary calorie allowances based on the 2005 U.S. Dietary Guidelines (2). For this reason, the American Heart Association Nutrition Committee recommended population-wide reductions in added sugars intake. The present statement from the American Heart Association and the American Diabetes Association addresses the potential role of nonnutritive sweeteners (NNS) in helping Americans to adhere to this recommendation in the context of current usage and health perspectives.

By definition, NNS, otherwise referred to as very low-calorie sweeteners, artificial sweeteners, noncaloric sweeteners, and intense sweeteners, have a higher intensity of sweetness per gram than caloric sweeteners such as sucrose, corn syrups, and fruit juice concentrates. As a caloric sweetener replacement, they are added in smaller quantities; hence, they provide no or few calories. In our current food supply, NNS are widely used in thousands of beverages and other food products such as diet soft drinks, yogurts, desserts, and gum. Food manufacturers often use a blend of NNS or use a blend of sugar and NNS to improve the flavor acceptability of NNS. In developing this scientific statement, the writing group reviewed issues pertaining to NNS in the context of data on consumer attitudes, consumption patterns, appetite, hunger and energy intake, body weight, and components of cardiometabolic syndrome. The objective was to review the literature to determine whether there were adequate data to provide guidance for the use of NNS.

The focus of the statement is on the 6 NNS that are described in Table 1. Aspartame, acesulfame-K, neotame, saccharin, and sucralose are regulated as food additives by the U.S. Food and Drug Administration and therefore had to be approved as safe before being marketed. Regarding stevia, at this time, the U.S. Food and Drug Administration has not made a determination as to the Generally Recognized As Safe status, but has issued no objection letters for a number of Generally Recognized As Safe notifications for stevia sweeteners (<http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASNotificationProgram/default.htm>). Because all 6 of these NNS have current U.S. Food and Drug Administration approval, issues related to safety of these compounds are not addressed. In addition, the review of the literature is primarily restricted to human studies in which noncaloric sweeteners are used as a replacement for caloric sweeteners.

# Literature Search

## Approach

The literature search was conducted by using the following search terms: nonnutritive sweetener(s), artificial sweetener(s), noncaloric sweetener(s), diet (beverages and soft drinks), and the names of each of the 6 NNS available for use in the United States (including their brand names). The initial search by use of PubMed was limited to original research published after 2000, studies conducted in humans (primarily restricted to controlled trials and prospective cohort studies), and systematic reviews. Additional articles were identified by use of the literature cited in the original publications and review articles, and the Evidence Analysis Library of the American Dietetic Association (<http://www.adaevidencelibrary.com/>), as well. A Google search was used to identify published marketing research related to consumer views of NNS.

Nonnutritive sweeteners (3–6)

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## Summary of available literature

The literature search yielded relatively few research studies that focused on the specific objective of this scientific statement, the potential role of NNS in facilitating reduction of added sugars intake in humans. This is likely attributable to inherent complexities involved in the design and implementation of these types of studies. In particular, experimental studies designed to test the effects of NNS on health outcomes in food are particularly challenging, because replacing added sugars with NNS alters dietary composition (i.e., assuming portion size is not reduced, the relative proportions of fat, protein, and carbohydrate may be changed or the nature of the carbohydrate alone may be changed) in ways that can add potential confounding to the simple comparison of added sugars versus NNS. The majority of the human data in prospective observational studies and randomized controlled trials on the use of NNS focus specifically on diet soft drinks as a replacement/displacement of regular soft drinks. Assessment in large prospective cohorts of the use of NNS from sources other than diet soft drinks has been limited methodologically by such factors as lack of specificity in intake data collected by food frequency questionnaires (FFQs) and lack of accurate NNS composition values in available databases. Much of the published literature on NNS addresses research conducted in animal models and evaluations of potential toxicity, neither of which were areas of focus for this statement. Hence, the review that follows is notably limited by the lack of an extensive evidence base.

# Consumer Attitudes and Rationale for Using or Avoiding NNS

Food and beverage industry publications provide some insights into consumers' rationale for NNS use. As expected, some of this use is tied to consumer efforts to decrease their intake of calories and caloric sweeteners. A 2012 survey of American adults by the International Food Information Council Foundation found that 51% of the respondents reported trying to limit sugar, 44% were trying to limit high-fructose corn syrup, and 29% were trying to limit low-calorie sweeteners (7). When asked to select ≥1 choices from a list of reasons for using NNS, 41% chose "are an option for people with diabetes," 41% chose "can reduce the calorie content of foods," 40% chose "can play a role in weight loss or weight management," 30% chose "can be part of an overall healthful diet," and 31% chose "don't know enough about them to provide an answer." Seventy-three percent of those consuming NNS reported doing so to reduce total calories consumed.

Although consumer demand for reduced-calorie and added-sugars products has resulted in a proliferation of new NNS products, American consumers have concerns about NNS, as well. In a Mintel survey conducted in a representative sample of Americans, the vast majority of survey respondents (81%) agreed with the statement, "Sugar-free foods do not taste as good as those made with real sugar" (7a). The Mintel survey also found that 64% of respondents indicated they were concerned about the safety of "artificial" sweeteners (7a). Survey data from the Hartman Group indicated that 44% of consumers reported they "deliberately avoid/reduce intake of artificial (flavors, sweeteners, colors or dyes)," but, when asked about specific NNS, the proportion drops to 39%, 33%, and 21% for saccharin, aspartame, and sucralose, respectively. The survey did not include other NNS (8). Consumers' concerns about taste and artificial ingredients may motivate food manufacturers to promote sucralose as being made from "real" sugar and stevia-derived NNS as being "natural" and made from the leaves of the stevia plant. Consumer education will need to stress that promotion of a certain NNS as "real" or "natural" does not necessarily indicate that such products are more efficacious or safer than other NNS.

# Estimates of Consumption Patterns

Information on the amounts of NNS in beverages and foods is limited and not easily accessible. Food processors and manufacturers are required to list the NNS in product ingredient lists but, with the exception of saccharin, are not required to provide the amount used in products or to release the information to federal agencies or the general public (3). Between 1999 and 2004, there was a large increase in the number of new products with NNS available in the United States, estimated to be \>6,000 (9). Of the 6 available NNS, sucralose (found in 2,500 products), acesulfame-K (found in 1,103 products), and aspartame (found in 974 products) were the most popular (9). NNS are most commonly used in carbonated beverages (3). Estimates of consumption patterns are typically made by reporting amounts of beverage and food items containing NNS, rather than amounts of specific NNS consumed.

## Nationwide Food Consumption Survey, National Health and Nutrition Examination Survey, andU.S. Department of Agriculture Economic Research Service Data

Mattes and Popkin (3) examined the trends in consumption of foods and beverages with NNS among Americans aged ≥2 years by use of the U.S. Department of Agriculture Nationwide Food Consumption Survey for 1965, 1977 to 1978, and 1989 to 1991 and the National Health and Nutrition Examination Survey (NHANES) for 1999 to 2000, 2001 to 2002, and 2003 to 2004. Consumption of NNS in both beverages and foods increased over time. The survey data indicate that, on any given day, 15% of the U.S. population consumed NNS in 2003 to 2004 in comparison with 3% in 1965. The proportion of the population choosing beverages with NNS remained relatively stable between 1989 and 2004 (10.1% and 10.8%, respectively), whereas the proportion consuming NNS in foods increased from 3.2% to 5.8% during that time period (3). Notably, the increase in NNS products was not accompanied by a corresponding decrease in reported consumption of products sweetened with added sugars, which indirectly suggests that NNS are not being used to displace products sweetened with added sugars. This same pattern of parallel increases in both NNS products and products with added sugars has been reported elsewhere (10,11) and is evident from per capita food availability data as compiled by U.S. Department of Agriculture Economic Research Service for regular versus diet soft drinks, with intake increasing primarily from 1980 to 1990 and then leveling off between 1990 and 2003 (Fig. 1).

One of the authors (L.M.S.) examined the prevalence of consuming NNS foods and beverages in children and adults with the use of data from NHANES 2007–2008. Dietary intake in NHANES was assessed by a 24-hour recall in-person interview, which presents a "snapshot" of foods and beverages consumed by NHANES participants on a given day*.* The most frequently reported and consumed NNS product was NNS beverages in comparison with intake of NNS foods or packets of artificial sweeteners among male and female children and adults in NHANES 2007–2008 (Fig. 2; L.M. Steffen, unpublished data, 2011). After further examination of NNS beverage intake by age group, the prevalence of consumption of these beverages increased across age groups, especially among females (Fig. 3; L.M. Steffen, unpublished data, 2011). In general, more adults were NNS beverage consumers than children or adolescents.

## Other perspectives on NNS users

Dietary intake assessed by FFQ, as is common in observational cohort studies, provides an estimate of those who "ever" versus "never" use NNS-containing beverage or food items included in the questionnaire; this is a different perspective than that derived from 24-hour recall data as above. The prevalence of saccharin intake in 1980 (the only NNS available at that time) was assessed in a subset of the Nurses' Health Study (NHS) characterized as being free from major illnesses and other lifestyle factors that might modify weight gain. Intake was assessed by use of a 61-item FFQ completed by 31,940 women. The report indicated that the prevalence of saccharin users was 56% (12). In the Health Professionals Follow-up Study (HPFS), artificially sweetened beverage intake was assessed in 1986 and every 4 years after that during 20 years of follow-up with use of a 131-item FFQ (13). By use of a cumulative average over this time period, 54% of the men reported consuming artificially sweetened beverages at least 2 times per month. The San Antonio Heart Study enrolled a randomly selected sample from non-Hispanic white and Mexican American neighborhoods. Among 3,682 participants who were examined first between 1979 and 1988 and then again 7 to 8 years later, 48% reported some consumption of NNS in beverages that included diet soft drinks, coffee, or tea. Consumption of NNS in food items was not assessed (14).

# Appetite, Hunger, and Energy Intake

For NNS-sweetened items to successfully contribute to reductions in calories from added sugars or other sources, they must also avoid causing compensatory energy intake immediately or later in the day. If choosing a diet soft drink over a regular calorie soft drink leads to a decrease of ∼100 kcal per 8-oz serving, but later in the day causes an alteration in appetite or hunger that results in an additional 50, 100, or 200 kcal of intake, the initial "calorie saving" effect would be altered or reversed. Or, if choosing a diet soft drink enables an individual to simultaneously justify eating a 150-calorie snack, the calorie savings from the NNS would also be negated.

Mattes and Popkin (3) critically examined the evidence for effects of NNS on compensatory appetite and food intake. The 8 potential mechanisms they reviewed are listed and briefly described in Table 2. The review concluded that the available evidence either refuted or was insufficient to refute or support each of these potential mechanisms or hypotheses for NNS increasing appetite, hunger, or energy intake (3).

Potential mechanisms of effects of NNS on compensatory appetite and food intake

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The complexity in attributing the extent to which NNS consumption is compensated for by intake of other foods is fraught with methodological challenges both in controlled feeding experiments and free-living trials. According to International Food Information Council Foundation research, \<10% of Americans can accurately estimate the number of calories they should consume in a day. Consumer education on daily energy requirements is needed, and that products containing NNS may assist in weight control when used in place of full-calorie products, if not compensated for otherwise. Controlled feeding experiments might or might not show an impact of NNS on future caloric intake, but, by definition, these are not real-world situations. Conversely, in the real world, so many factors impact food choice that attributing caloric intake after NNS consumption uniquely to that consumption may also not be true and may be confounded particularly by reverse causality; that is, the previous NNS choice might be deliberate in anticipation of a known future exposure to higher caloric meal.

## Energy intake compensation

Various studies that have examined energy intake as a primary or secondary outcome have used designs that contrast beverages and foods made with caloric sweeteners versus NNS. In a meta-analysis of weight loss studies that tested the effectiveness of aspartame in reducing energy intake, data from 12 studies were pooled to address energy intake compensation (15). The weighted average of energy intake compensation in the ∼24 hours that followed aspartame intake was 32% (i.e., 68% of the original energy deficit was uncompensated and was maintained through the subsequent period of intake). Of the 4 studies from the meta-analysis that used beverages alone, the compensation was just 15% for the subsequent 24 hours; that is, the data suggest there is less compensation in beverages than foods, resulting in a more effective net reduction in calories when replacing sweetened beverages with NNS beverages (15). The implication here is that anything \<100% compensation results in a net reduction in 24-hour energy intake.

One of the studies included in the meta-analysis involved 24 men and women who consumed 4 different beverages, each for 4 weeks, in a crossover design. Subsequent food intake was monitored. The beverages included a full caloric beverage (sucrose) versus an NNS beverage (aspartame), provided with either an orange or raspberry flavor. Data were collected at baseline and after 4 weeks of habituation (16). The investigators observed a significant lack of energy intake compensation over the course of the day among study participants when they were consuming the full caloric beverages relative to the NNS beverages. In another study, 21 overweight adults were assigned to consume 28% of their energy intake as sucrose, mostly as beverages. After 10 weeks, there was a significant increase in their energy intake, body weight, fat mass, and blood pressure (17). These effects were not observed in a similar group of 20 overweight adults who consumed NNS in place of the sucrose, but otherwise had a similar dietary intake composition in comparison with the other group (17). In a controlled feeding experiment that involved a preload snack containing sucrose (493 kcal), aspartame (290 kcal), or stevia (290 kcal), the energy intake of subsequent meals was similar; the energy intake for the combined preload snack and later meals combined was lower for the aspartame and stevia phases versus the sucrose phase (*P* = 0.01) (18). All of these studies, therefore, observed a net reduction in energy intake for NNS use relative to the comparison condition with sucrose.

Not all such studies have observed a net reduction in overall energy intake when NNS were used instead of caloric sweeteners. A 4-week trial, providing either diet soft drinks (sweetened with aspartame) or regular calorie soft drinks resulted in similar total energy intake in healthy-weight adults of both sexes (19) and in overweight women as well (20). Similarly, the results of a 25-week study conducted in adolescents suggested that normal-weight youth compensated for the energy reduction when consuming diet beverages. However, as evidence of the complexity of this issue, the same study reported that the use of NNS beverages did result in a net energy deficit in those adolescents in the highest body mass index (BMI) tertiles (a conclusion that should be tempered by awareness that the intervention group was counseled to replace sugar-sweetened beverages with diet beverages and/or water, making it difficult to differentiate the effects of NNS versus water) (21).

Overall, the limited human studies literature in this area of NNS research suggests that compensation is an important factor to consider when assessing impact on energy intake over periods of ≥24 hours. Further studies are warranted to address some of the complexities inherent in this issue, including the potential impact of being informed versus naïve about the presence or absence of NNS in items being consumed. It is worth noting that, in preschool children, when energy intake is reduced by substitution of nutrient-dense, lower-calorie food choices instead of NNS (e.g., fruits and vegetables), some energy intake compensation occurs, but net energy intake remains lower over several days (22,23).

## Appetite, or preference, for sweet taste

Examination of the potential effects of NNS on appetite requires consideration of genetic and environmental factors that may regulate preference for intensity of sweet taste (24–26). One focus of such research concerns the metabolic/hormonal responses to both caloric sweeteners and NNS to determine whether these can be linked to mechanisms that promote future weight gain or other adverse physiological consequences. Sweet taste is mediated largely by a single receptor composed of the 2 subunits, TAS1R2 and TAS1R3. Understanding how the polymorphism of this receptor gene affects preferences is important to understanding usage of NNS. Recent data in mice suggest that additional mechanisms may exist, but these have not been confirmed in humans (27). Newer animal research also suggests the possibility that sweet-taste receptors exist in the gut, triggering complex metabolic/hormonal responses to NNS. The impact of these receptors and their metabolic consequences on obesity and diabetes mellitus in humans is just beginning to be studied (28). Although ingestion of foods containing NNS may trigger a wide array of responses related to food absorption that may impact future food consumption (29), adverse effects related to these gut receptors have yet to be identified (30,31).

A preference for higher intensity of sweetness exists in humans in infancy, declines in adolescence in comparison with infancy, and then plateaus or slowly declines in young adulthood, and may or may not decline further in old age (32,33). There is significant interindividual variation in sweet- and bitter-taste preference, and this preference may be genetically determined (34–37). These genetic differences may impact preferences for both caloric sweeteners and NNS, which may also have a bitter-taste component. Although animal research suggests that sweet-taste preference can be conditioned through repeated exposure (38), preference for sweet taste in humans is more complex and involves many central receptor mechanisms, including but not limited to dopamine signaling (39). Critical factors such as food culture and social expectations may also have an impact (3). The presence of caloric sweeteners may also enhance taste acceptance (40).

# Cardiovascular and Metabolic Health Outcomes

The potential effects of NNS on body weight, cardiometabolic variables, and diabetes mellitus/glycemic response were examined. The evidence considered was limited to prospective studies and controlled trials conducted in humans. For the controlled trials, primary emphasis was given to study designs that involved replacing caloric sweeteners with NNS. Prospective cohort studies were also considered, but cross-sectional data were not considered. Although NNS intake may precede and lead to changes in weight or metabolic variables, the opposite is also plausible (i.e., individuals who become overweight/obese may seek out NNS items after becoming overweight/obese or being informed they have type 2 diabetes mellitus). Therefore, it is not possible to distinguish the temporal relationship between these by using cross-sectional studies, and, as will be discussed, it can be difficult to distinguish this relationship even in some prospective studies.

## Body weight

The vast majority of data available related to NNS consumption and body weight has focused on creating an energy deficit by substituting NNS for sugars in the beverage component of the diet, with only a few intervention studies also targeting foods. Findings in both controlled intervention trials and prospective observational studies have been inconsistent.

Controlled intervention trials have reported that use of products with NNS results in weight loss or has negligible effects on weight. In a review of 16 trials with a primary focus on aspartame and weight control, weight loss was a primary outcome in 9 of the 16 trials (15). Beyond summarizing the overall pattern of success with weight loss in these studies, the investigators attempted to quantify the magnitude of caloric substitution required for meaningful weight loss. By their calculations, a weight loss of 0.2 kg/week over 12 weeks was achieved from a net energy deficit of 220 kcal/day attributable to aspartame substitutions for caloric sweeteners. Determining the net energy deficit required consideration of energy intake compensation, which was different for foods and beverages (as described previously, see energy intake compensation). For foods it was determined that displacing 330 kcal/day of sugar (82.5 g), and assuming 32% compensation, would yield the 220-kcal deficit. For beverages, with a calculated 15% compensation, 260 kcal of caloric sweetener, or 70 g (the amount found in ∼2, 12-oz soft drinks), would need to be displaced to yield the same net 220-kcal deficit.

In a study conducted with 103 subjects who were 13 to 18 years of age and who regularly consumed sugar-sweetened beverages, BMI change was the primary outcome. The randomized trial contrasted home delivery of NNS beverages (i.e., water and diet soft drinks, iced teas, lemonade, and punches) to a control group that did not receive beverages and were instructed to continue their habitual consumption. Overall, there were no significant differences in BMI changes over the 25-week period; however, in a post hoc stratified analysis of participants in the highest tertile of baseline BMI, the BMI change was significantly greater, reflecting weight loss, in the group receiving the NNS beverages (21). Another study involved a family-based randomized trial testing the impact of promoting sucralose and sucralose-containing products as substitutes for caloric sweeteners in beverages and foods (41). This study was conducted in conjunction with instructions to add 2,000 steps/day to their habitual physical activity. One hundred families assigned to the intervention were compared with 93 families assigned to follow their habitual diet and activity patterns. After 6 months, none of the primary study outcomes (changes in BMI for age, waist circumference, or percentage of body fat) were significantly different between treatment groups, either for the children (primary target population), or their parents (secondary target population). In post hoc analyses, the investigators found a borderline significant improvement in the intervention group for the percentage of children who maintained or reduced their BMI for age.

As would be expected, controlled intervention trials involving dietary manipulations, as described above, are generally limited to relatively short durations of ≤6 months (1 exception being a 16-week intervention with aspartame-sweetened foods and beverages with a 1-year maintenance period and 2 years of follow-up; this study was included in the review of 16 trials previously described) (15,42). Longer-term perspectives are available from prospective observational studies, with the important limitation that these studies can address only associations, and not causal effects.

A limited number of prospective observational studies have addressed NNS and weight, most of which assessed NNS intake by FFQ. None of the studies were designed specifically to assess associations between NNS and weight. Two studies published \>20 years ago reported a positive association between NNS use and weight gain (12,43). However, these findings were observed by the use of limited dietary assessment methods, and in selected (i.e., not representative) study populations. A 2008 study examining the San Antonio Heart Study population reported a positive dose-response association between artificially sweetened beverage consumption (i.e., diet soft drinks, and coffee and tea with added NNS) and incidence of overweight/obesity during a 6- to 7-year follow-up period of 5,158 adults. The association was consistent across sex and ethnicity; overall, the change in BMI was 47% greater among NNS users than nonusers after adjusting for baseline BMI and demographic and behavioral characteristics (14). A 2011 study reported findings on associations of a broad range of diet and lifestyle variables with 12- to 20-year weight change observed in 3 separate cohorts that included 120,877 U.S. women and men from the NHS, NHS II, and the HPFS. For a given unit of 1 diet soft drink serving/day, the association with weight change was not statistically significant in the NHS, whereas there was a statistically significant but modest association with weight loss (\<0.25 lb/4 years) observed in NHS II and HPFS after adjusting for a broad range of demographic and lifestyle variables (44).

A separate publication based on only the HPFS population focused specifically on sugar-sweetened beverage versus artificially sweetened beverage (ASB) intake and provides important insights into the opportunities and limitations of the perspective offered by prospective observational studies. Cumulative averages of intake were determined by FFQs administered every 4 years for 20 years, starting in 1986, and the main outcome of interest in this publication was incidence of type 2 diabetes mellitus. At the start of the study, in 1986, participants were asked to report any weight change in the preceding 5 years, between 1981 and 1986. The weight change data were presented separately for those who reported losing weight (14%–18% of the population, depending on the specific quartile of ASB intake \[L. de Koning, personal communication, 2011\]) and those who reported gaining weight (45%–51% of the population, depending on the specific quartile of ASB intake) in that time frame; the remainder reported no weight change (13). Among those who lost weight, there was a significant trend of greater weight lost for higher versus lower ASB intake quartiles (0.9 ± 0.2 versus 0.5 ± 0.5 kg, means ± SD, respectively, for the highest versus lowest quartile). Among those who gained weight, there was a significant trend of greater weight gained for higher versus lower ASB intake quartiles (2.6 ± 4.6 versus 1.8 ± 3.4 kg, respectively). This may appear contradictory; how could greater intake be associated with both greater weight loss and greater weight gain? However, this suggests there may be importantly different subsets of the population: one that successfully uses ASB for weight control and another that does not. On the other hand, the different time frames of the weight change data versus ASB intake data must also be considered; the 1981 to 1986 weight change preceded the 1986 to 2006 cumulative average ASB intake reported in the article; from a chronological perspective it is the weight change that is predicting ASB consumption, rather than vice versa (i.e., weight gain may predict subsequent increased ASB consumption). Three additional findings presented in this study further highlight the complexities involved: the following trends for baseline characteristics for the full study population, regardless of 1981 to 1986 weight change, were all statistically significantly related to ASB intake; diet quality as assessed by the Alternative Healthy Eating Index was higher, total energy intake was lower, and BMI was higher for those consuming greater amounts of ASB. Overall, the data from this study suggest there may be important subsets of the population that incorporate NNS products into their diet with different objectives or with differential success, and that there is likely substantial confounding in these types of analyses given the many dietary and health variables that correlate with NNS intake (12,43).

### Assimilating results from intervention and observational studies.

The 2 sets of studies reviewed above help to demonstrate what have been and will continue to be challenges in reaching definitive conclusions about the effectiveness of NNS in supporting energy intake restriction and promoting weight loss and weight control. The short-term nature of intervention studies limits their ability to observe long-term weight change, and the typical personal/individual support from study staff and related heightened awareness of NNS intake among study participants limit the generalizability of the findings. On the other hand, among the observational studies, the challenges of accurately assessing NNS intake, the many potential confounders, and the difficulty in determining directionality (e.g., possible reverse causality) represent important limitations. Nonetheless, the strength of intervention trials in establishing causality and the opportunity in observational studies to address long-term changes in health outcomes suggest that both approaches will continue to be useful and complementary in providing guidance regarding NNS use. Currently, available data provide, at best, modest support for a modest effect on weight with NNS use, with many important caveats to the available published findings and ample opportunity for future studies to build on past studies to more effectively address this issue.

## Cardiovascular disease risk factors, cardiometabolic syndrome, and coronary heart disease

Several prospective studies have been conducted to examine associations of NNS soft drink (i.e., diet soft drink) intake with chronic disease, including coronary heart disease (CHD), chronic kidney disease, and cardiometabolic syndrome and its individual components. In the NHS, consuming ≥2 servings of diet soft drinks per day was associated with an increased risk of CHD and chronic kidney disease in comparison with consuming \<1 serving of diet soft drinks per month over 11 to 12 years of follow-up (45,46). Middle-aged men and women consuming \>1 servings of diet soft drinks per day had \>30% higher risk of developing metabolic syndrome over 9 years of follow-up (47). Similar results were also observed for the components of metabolic syndrome (47). In the Northern Manhattan Study of \>2,500 multiethnic adults with 10 years of follow-up and a cardiovascular event incidence of ∼2%, daily diet soft drink intake was associated with an increased risk of events; regular soft drink intake was not associated with events. However, the small numbers of events, the unusually small numbers of daily consumers of diet soft drinks in this population, and the plausibility of reverse causality led the authors to caution against possible misinterpretation of the findings (48). In contrast, a recent report from the HPFS examining CHD incidence over 22 years in almost 43,000 men with 3,683 CHD events, observed a positive association between sugar-sweetened beverage intake and CHD risk, but no association with ASB (49). The authors specifically noted in their discussion that ASB consumption was probably, at least in part, attributable to response to the diagnosis of a chronic condition. Finally, in a recent study of the joint associations of consuming a prudent (healthy) or Western diet pattern with or without diet beverage consumption among young adults with \>20 years of follow-up (CARDIA \[Coronary Artery Risk Development In Young Adults\] study), consumers of both a prudent (healthy) diet pattern and diet beverages had a lower risk of having elevated glucose or low high-density lipoprotein-cholesterol, but not the metabolic syndrome, than nonconsumers following a prudent diet pattern or consumers and nonconsumers following a Western diet pattern (50). In general, the literature in this area is sparse, limited to prospective observational studies, and examines NNS intake from beverages, but not foods. In addition, these observational prospective studies can only identify associations, not causal determinants, and there is often a strong plausibility of reverse causality for some of the significant associations observed.

## Diabetes mellitus and glycemic response

A recent publication of 20-year follow-up data from healthy men in the HPFS addressed the relationship between intake of sugar-sweetened beverages and ASB and the development of type 2 diabetes mellitus (13). For sugar-sweetened beverages, the association with developing diabetes mellitus remained significant after adjusting for potential confounders. ASB intake was significantly associated with developing type 2 diabetes mellitus in an age-adjusted analysis. However, the association was no longer significant in subsequent multivariate-adjusted analysis; the association between ASB and diabetes mellitus was largely explained by health status/family history related to diabetes mellitus risk, preenrollment weight change, dieting, and BMI. These findings suggest that the men who consumed ABS may have done so to address a weight gain and health conditions associated with increased risk of developing diabetes mellitus.

With regard to NNS and glycemic response, 4 randomized trials that varied from 1 to 16 weeks in duration found no significant difference between the effects of NNS and various comparisons (sucrose, starch, or placebo) on standard measures of glycemic response (i.e., plasma glucose and insulin, HbA~1c~, C-peptide) and, in general, did not detect clinically relevant effects (51–54). One study that involved contrasting treatment arms assigned participants to either *1*) a diet consistent with American Diabetes Association guidelines (control arm) or *2*) a lower calorie experimental diet containing sucralose. The result was greater reductions in HbA~1c~, weight, and BMI for the experimental diet group (55). The experimental diet, however, differed from the control diet in fat replacer and fructose content, and sucralose content, as well, and therefore it is not possible to determine the separate contribution of the sucralose content to the study effects. The submission to the U.S. Food and Drug Administration to designate stevia extract, rebaudioside A, also known as rebiana, with Generally Recognized As Safe status noted the lack of effects on blood glucose. Information about a lack of blood glucose effects was presented to address claims that some stevia-derived compounds had a therapeutic blood glucose–lowering benefit (56). Potential effects of beverages or foods containing NNS on glycemic response are likely attributable to the replacement of carbohydrate by NNS rather than any direct effect. The American Dietetic Association (renamed the Academy of Nutrition and Dietetics in 2012) concluded in a 2010 review that, in general, NNS do not affect glycemic response (do not raise postmeal glucose response or have a negligible inherent effect on glycemic response) in people with diabetes mellitus. Although the intrinsic glycemic effects of NNS are similar to other calorie-free/carbohydrate-free products, some NNS-containing products may contain energy and carbohydrate from other sources that need to be considered (57).

# Needs for Further Research

Because of the paucity of well-designed human studies that address the specific, practical, public health issues, as presented above, further research is warranted in several areas.

## NNS and energy compensation

The most obvious potential benefit of NNS is the substitution of energy from added sugars, thereby facilitating both energy and carbohydrate intake reductions (with potential glycemic control benefits). However, what would appear to be a potential benefit is complicated by compensation; that is, the extent to which an immediate energy and carbohydrate reduction through NNS use is followed by a subsequent increase in energy intake (regardless of macronutrient type) beyond what would have been consumed in the absence of the use of NNS. Existing data suggest that there is partial compensation with differences in intake after consuming food and beverages containing NNS. Energy intake compensation appears to be greater after consuming foods containing NNS than that for beverages containing NNS. Future work should test and compare different food and beverage vehicles of the NNS, and compare the different NNS themselves and NNS combinations as well, in an effort to maximize potential impact.

## NNS mechanisms of action and pathophysiology

Research in the area of energy compensation would be facilitated and complemented by continued research into the mechanisms of potential NNS effects. This would include, but not be limited to, increasing the body of knowledge of oral, gut, and neural receptors, and hormonal responses that contribute to alterations in food preferences, appetite regulation, and satiety that are elicited by NNS. This research should inform the selection of foods, beverages, specific NNS (or combinations), and perhaps specific population subsets for studies of compensation effects.

## Randomized controlled trials using NNS to impact body weight or glycemic control

Well-designed and controlled designed clinical trials are needed to determine the long-term effect of NNS on body weight regulation and glycemic control at different points in the life cycle. Weight control studies (e.g., weight loss) should be minimally 1 year in duration or longer, whereas glycemic control studies could be shorter in duration. Studies of both end points would benefit from better informed selections of the types, amounts, combinations, and vehicles of NNS delivery, as described in the previous 2 areas of suggested further research.

## Assessment of NNS intake

Immediate needs include updating of current food databases with accurate data for the NNS content of foods and revision of instruments used to estimate food intake to accurately capture NNS content.

# Conclusions and Recommendations

At this time, there are insufficient data to determine conclusively whether the use of NNS to displace caloric sweeteners in beverages and foods reduces added sugars or carbohydrate intakes, or benefits appetite, energy balance, body weight, or cardiometabolic risk factors. Limiting added sugars is an important strategy for supporting optimal nutrition and healthy weights, as concluded in the 2009 American Heart Association scientific statement "Dietary Sugars Intake and Cardiovascular Health" (1). Monitoring carbohydrate intake, which includes limiting added sugars, is also a key strategy to achieve glycemic control as published in the American Diabetes Association clinical practice recommendations (58). There are some data to suggest that NNS may be used in a structured diet to replace sources of added sugars and that this substitution may result in modest energy intake reductions and weight loss. Successful reduction in energy intake requires that there is incomplete compensation of energy reduction from the use of NNS-containing beverages and/or foods. The impact of incorporating NNS and NNS-containing beverages and foods on overall diet quality should be included in assessing the overall balance of benefits and risks. Apparent from the available literature is the paucity of data from well-designed human trials exploring the potential role of NNS in achieving and maintaining a healthy body weight and minimizing cardiometabolic risk factors.

The evidence reviewed suggests that when used judiciously, NNS could facilitate reductions in added sugars intake, thereby resulting in decreased total energy and weight loss/weight control, and promoting beneficial effects on related metabolic parameters. However, these potential benefits will not be fully realized if there is a compensatory increase in energy intake from other sources.

# Disclosures

Writing group disclosures

![](1798tbl3)

Reviewer disclosures

![](1798tbl4)

## Acknowledgments

For potential conflicts of interest, please see DISCLOSURES.

The authors thank Xia Zhou, MS, for programming, data presented in Figs. 2 and 3, and working with Dr. Steffen.

# References

[^1]: The American Heart Association and the American Diabetes Association make every effort to avoid any actual or potential conflicts of interest that may arise as a result of an outside relationship or a personal, professional, or business interest of a member of the writing panel. Specifically, all members of the writing group are required to complete and submit a Disclosure Questionnaire showing all such relationships that might be perceived as real or potential conflicts of interest.

[^2]: This document was approved by the American Heart Association Science Advisory and Coordinating Committee on April 18, 2012, and by the American Diabetes Association Executive Committee on April 19, 2012.

[^3]: Correspondence about this article can be directed to [firstname.lastname@example.com](firstname.lastname@example.com).

[^4]: This article has been copublished in *Circulation*.

[^5]: © 2012 by the American Heart Association, Inc., and the American Diabetes Association.


author: Philip E Bourne; Leo M Chalupa\* To whom correspondence should be addressed. E-mail: <firstname.lastname@example.com>[^1]
date: 2006-02
references:
title: Ten Simple Rules for Getting Grants

This piece follows an earlier Editorial, "Ten Simple Rules for Getting Published" \[1\], which has generated significant interest, is well read, and continues to generate a variety of positive comments. That Editorial was aimed at students in the early stages of a life of scientific paper writing. This interest has prompted us to try to help scientists in making the next academic career step—becoming a young principal investigator. Leo Chalupa has joined us in putting together ten simple rules for getting grants, based on our many collective years of writing both successful and unsuccessful grants. While our grant writing efforts have been aimed mainly at United States government funding agencies, we believe the rules presented here are generic, transcending funding institutions and national boundaries.

At the present time, US funding is frequently below 10% for a given grant program. Today, more than ever, we need all the help we can get in writing successful grant proposals. We hope you find these rules useful in reaching your research career goals.

# Rule 1: Be Novel, but Not Too Novel

Good science begins with new and fresh ideas. The grant writing process should be a pleasure (no, we are not kidding), for it allows you to articulate those ideas to peers who have to read your grants but not necessarily your papers. Look at grant writing as an opportunity to have an impact. Feel passionate about what you are writing—if you are not passionate about the work, it is probably not a good grant and is unlikely to get funded. "Me-too" science will not get funded when funding levels are low. On the other hand, science that is too speculative will not be supported either, particularly when funds are tight—sad but true.

# Rule 2: Include the Appropriate Background and Preliminary Data as Required

You need to convince reviewers that the work you propose needs to be done and that you are the best person to do it. Different granting programs require differing amounts of preliminary data. For certain programs, it can be said that the work must be essentially done before the grant is awarded, and that the funds are then used for the next phase of the research program. There is some truth in this. So where appropriate, do provide some tantalizing preliminary result, making sure to tell the reviewers what these results imply with respect to the specific aims of your proposal. In formulating the motivation for your proposal, make sure to cite all relevant work—there is nothing worse than not appropriately citing the work of a reviewer! Finally, convince the reviewer that you have the technical and scientific background to perform the work as proposed.

# Rule 3: Find the Appropriate Funding Mechanism, Read the Associated Request for Applications Very Carefully, and Respond Specifically to the Request

Most funding organizations have specific staff to assist in finding funding opportunities, and most funding agencies have components of their Web sites designed to help investigators find the appropriate programs. Remember, programs want to give away money—the jobs of the program's staff depend on it. The program staff can help you identify the best opportunities. If your grant does not fit a particular program, save your time and energy, and apply elsewhere, where there is a better programmatic fit.

# Rule 4: Follow the Guidelines for Submission Very Carefully and Comply

Many funding bodies will immediately triage grants that do not comply with the guidelines—it saves the program time and money. This extends to all the onerous supporting material—budget justification, bibliographies, etc. Get them right and keep them updated for future applications. Even if it goes to review, an inappropriately formulated application may aggravate the reviewers, and will have a negative impact even if the science is sound. Length and format are the most frequent offenders.

# Rule 5: Obey the Three Cs—Concise, Clear, and Complete

The grant does not have to fill the allotted page count. Your goal should be to provide a complete reckoning of what is to be done, as briefly as possible. Do not rely on supplements (which may not be allowed) or on Web sites (review may be actively discouraged since it has the potential to compromise anonymity). Specify the scope up-front and make sure it is realistic with respect to the funds requested. A common temptation for inexperienced grant writers is to propose to do too much. Such applications are usually judged as overly ambitious and consequently poorly rated.

# Rule 6: Remember, Reviewers Are People, Too

Typically, reviewers will have a large number of grants to review in a short period. They will easily lose concentration and miss key points of your proposal if these are buried in an overly lengthy or difficult-to-read document. Also, more than likely, not all the reviewers will be experts in your discipline. It is a skill to capture the interest of experts and nonexperts alike. Develop that skill. Unlike a paper, a grant provides more opportunity to apply literary skills. Historical perspectives, human interest, and humor can all be used judiciously in grants to good effect. Use formatting tricks (without disobeying rule 4), for example, underlining, bolding, etc., and restate your key points as appropriate. Each section can start with a summary of the key points.

# Rule 7: Timing and Internal Review Are Important

Give yourself the appropriate lead time. We all have different approaches to deadlines. Ideally, you should complete a draft, leave sufficient time to get feedback from colleagues, and then look at the grant again yourself with a fresh eye. Having a spectrum of scientific colleagues who are similar to the likely reviewer pool critique your grant is very valuable.

# Rule 8: Know Your Grant Administrator at the Institution Funding Your Grant

At the end of the day, this person is your best advocate. How well you understand each other can make a difference. Many grant administrators have some measure (limited to complete) discretionary control over what they fund. The more they know and understand you and your work, the better your chances of success. Do not rely just on E-mail to get to know the grant administrator. Do not be intimidated. Talk to them on the telephone and at meetings where possible—they want to help.

# Rule 9: Become a Grant Reviewer Early in Your Career

Being on review panels will help you write better grants. Understanding why grants get triaged before complete review, how a panel reacts to a grant, what the discretionary role of program officers is, and what the role of oversight councils is provide valuable lessons for writing successful grants of your own and for giving others advice about this process.

# Rule 10: Accept Rejection and Deal with It Appropriately

Rejection is inevitable, even for very good grants when funding levels are low. Learn to live with rejection and to respond appropriately. Do not be defensive; address each criticism head on and respond with facts and not emotional arguments. When resubmission is necessary, make it very clear to the reviewer that you understand what was wrong the first time. Indicate precisely how you have fixed the problems. In the resubmitted application, never argue with the validity of the prior review. If the grant was close to being funded the first time around, remind the reviewers of that fact by including the previous score if appropriate, and make it crystal clear why this version is much improved.

There are no previously unrevealed secrets to grant writing presented here. Rather, it is a concise picture intended to help our early career readers take the next step. If you feel like you need more detail, take a look at Kraicer's article \[2\]. Good luck on getting those grants.

# References

[^1]: Philip E. Bourne is a professor in the Department of Pharmacology, University of California San Diego, La Jolla, California, United States of America, and is Editor-in-Chief of *PLoS Computational Biology*. Leo M. Chalupa is a professor and chair in the Section of Neurobiology, Physiology, and Behavior, University of California Davis, Davis, California, United States of America.


abstract: A combination of mathematical modeling and direct observation of the swimming behavior of dolphin mother-calf pairs has shown how the calf can gain much of the energy required for swimming if it is positioned correctly relative to the mother, a situation that may be disrupted during the chases that result from tuna-fishing practices.
author: Pete Moore
date: 2004
references:
title: Examining dolphin hydrodynamics provides clues to calf-loss during tuna fishing

Anyone who has bought a can of 'dolphin-friendly' tuna knows that dolphins can be unintended victims of the tuna-fishing industry. Now, a mathematical model shows that dolphin calves can gain up to 90% of the energy required for swimming if they position themselves correctly relative to their mothers, but that the calves are in danger of being lost if this positioning breaks down \[1\]. As observations of dolphins in the wild support the model (see 'The bottom line' box for a summary of the findings), the new work starts to give a possible explanation why dolphin numbers remain at unnaturally low levels despite changes in the tuna-fishing industry aimed at protecting dolphin populations.

# A brief history of tuna and dolphins

During the 1970s an estimated 350,000 dolphins were killed each year as an unintended but inevitable **by-catch** of the tuna fishing in the eastern tropical Pacific Ocean (see the 'Background' box). This occurred because between 1959 and 1963 the tuna-fishing fleet switched from pole and line fishing to a technique using vast **'purse seine'** nets. Dolphins are caught because fishermen have known for a long time that tuna gather beneath schools of dolphins and very often seabirds congregate overhead - if you want to catch tuna, look for dolphins.

Purse-seine fisheries involve a fast moving boat and a helicopter that search for the telltale signs of seabirds and dolphins. On arriving near a school of dolphins the main boat launches four to six speedboats that operate like sheep dogs, chasing the dolphins so that they are kept together while the main boat encircles them with a one-mile long, 400-foot deep curtain of net, the top edge of which is held on the surface by floats (Figure 1). The chase tends to last between 15 and 30 minutes, but occasionally extends to up to an hour. Once the circle is complete, the fishermen pull a rope that draws the bottom of the net together. This captures the tuna, but also encloses the dolphins. Without assistance, the dolphins can get caught up in the net and, unable to reach the surface, drown.

Since the 1980s various changes in fishing procedures have reduced dolphin mortality. Before pulling the net in, the main boat turns and, because of the way it is constructed, the circular net forms a finger-like shoot opposite the large boat. The speedboats head for this part of the net and hold it open so that the dolphins can swim out. Given that dolphins experience this procedure fairly frequently, they appear to learn what to do. Their escape is helped by the fact that the net in this area is made of a very fine mesh so that they don't get caught, and divers swim inside the net actively pulling some of the stragglers out to the open ocean.

While the effort given to helping dolphins escape has cut deaths significantly, conservationists have been puzzled to note that dolphin numbers are nevertheless failing to recover. One possible clue to the problem came when researchers examining data collected by observers placed with the tuna fleet found that more lactating mothers were killed within the net than nursing calves. Elizabeth Edwards, a marine biologist working for the National Marine Fisheries Service in San Diego, USA, notes that "70-80% of lactating females killed were not killed with a calf, and the findings were consistent over several years". Edwards and her colleagues started looking to see whether calves could become separated from their mothers during fishing operations \[2,3\]. This would not be recorded in mortality statistics, but could radically influence the recovery of the dolphin population. "Dolphins are very precocious animals," says Frank Fish of West Chester University, in West Chester PA. "We always marvel at wildebeest and other ungulates that start moving with the herd within 20 minutes of being born, but dolphins are moving constantly, so the neonate doesn't even have the luxury of 20 minutes."

Marine biologists studying dolphin behavior frequently see mothers and calves swimming closely together at speeds of up to 2.4 m/sec, with young calves making little if any obvious swimming motions. The assumption is that the calf is **drafting** in its mother's **slipstream**, just as cyclists save energy by packing together during a race. The principle is widespread in biology. "We are always amazed at how smart animals are in their ability to conserve energy - such as ducklings' use of the flow pattern in the water that the mother produces as she swims along, so that they can reduce the amount of energy they need to expend to maintain that speed," says Fish. But in the case of dolphins there has never been any hard science aimed at seeing if this is a lovely myth or a physical reality.

Enter mathematician and aerodynamicist Daniel Weihs, who works at the Technion, Israel Institute of Technology, Haifa. He was involved in research some 25 years ago that aimed to find the speed dolphins needed to reach before they started jumping \[4\], and now he came back to the topic, looking for mathematical evidence of forces between mothers and calves (see the 'Behind the scenes' box for more of the rationale of the work).

# Reduce complexity

The first task in making a mathematical model of a biological situation is to identify the dominant effects and simplify the issue. Once calculations are made in this simplified world it is relatively easy to add back the complications. Weihs started by assuming that dolphins are ellipsoidal in shape and that their bodies are six times as long as they are wide. Using a regular symmetrical object makes the equations much less complex, and previous studies had indicated that dolphin bodies were closely represented by an ellipsoid. On top of this, people working on torpedoes and other underwater weaponry had already developed equations that predict ellipsoid behavior in water.

Next, he assumed that the water is inviscid - that is, it has no viscosity. This enabled him to employ d'Alembert's paradox. Named after eighteenth century French mathematician and physicist Jean le Rond d'Alembert, this paradox states that objects surrounded by an inviscid, incompressible liquid experience no drag as the liquid moves past. In doing this he had placed his theoretical dolphins into a simple Newtonian world where objects continue to move until influenced by some external force. "The main point here is that any single body under the conditions we are talking about produces no forces at all if it is moving at constant speed," explains Weihs. The question then is, what forces occur in this environment when you have two objects moving closely together?

Finally, he introduced two more assumptions: first, that the body shapes did not change during swimming; and second, that the dolphins were swimming at sufficient depth that they were not creating any surface waves. The results were clear. As the mother's body moves through the water, it pushes water sideways and outwards, and after she has moved forwards the water flows back in to fill the vacated space. "This means that if a second body is placed close enough to the first and within the area of forward motion of water, the relative velocity it feels will be less than its absolute velocity," notes Weihs.

Weihs' initial model showed that there were areas immediately in front of the mother dolphin, as well as to the side and behind, where a baby dolphin could swim and gain up to 90% of the force needed to move through the water from its mother. What's more, there were areas where Bernoulli attraction would actively pull a calf sideways, towards its mother.

# Add in some reality

Life, however, is not that simple. Any baby dolphin that tried to stay immediately ahead of its mother would have great difficulty. "The situation," explains Weihs, "would be akin to pushing a string and expecting it to stay straight - it would be too unstable." Again, being immediately behind the mother wouldn't work, because a real dolphin moves by forcing a jet of water backwards, by flexing its body and using its tail flukes. While the initial calculations might suggest that immediately behind the mother was a great place, common sense shows that it wouldn't. At the same time, the flexing of the mother's body means that any zone directly above or below her would be too unstable to be helpful to a calf. This leaves the two zones on either side of the mother and about two thirds of the way back as the two most stable and energetically favorable places for a youngster to hitch a lift.

Away from the mathematician's calculations, real dolphins also experience drag from the viscosity of the water. Adding this back makes the energy saving even more extreme. Any drag is proportional to the square of velocity, so positioning yourself in an area of forward-moving water allows you to experience much less drag. The energy required to achieve this velocity also increases as the cube of velocity. Consequently, any 10% saving in apparent velocity leads to a nearly 30% saving in energy. "The predicted force reduction is so substantial that it must play a major role in determining the calf's swimming position in practice," says Timothy Pedley, G. I. Taylor Professor of Fluid Mechanics in the Department of Applied Mathematics and Theoretical Physics at Cambridge, UK.

Dolphins do also rise to the surface and create surface waves. The energy used to create these waves constitutes another drag on the animals. Marine engineers calculate this as the Froude number, a non-dimensional coefficient defined as the ratio of inertial force to gravitational force. Applying this to the dolphins' situation reduces the effect of the previously mentioned gains. Consequently, swimming near to the surface presents added problems to a calf that is trying to keep up with its mother. The point of interest for the tuna-fishing scenario is that in chase situations dolphins need to be nearer to the surface, as they need to breathe more frequently.

Having determined the hydrodynamic basis for drafting, Weihs then applied his calculations to photographs taken from helicopters of mother-calf dolphin pairs swimming together in the wild. His conclusion is that in this real-life situation the calves gain between 30% and 60% of the force required to keep up with their mothers \[1\]. "This is a very nice model and is almost certainly the explanation for the observations of drafting," comments Pedley, adding: "it is not often that one can be so sure of the relevance of a mathematical calculation to biological phenomena."

# Energetic advantage

"Weihs' paper establishes the basis for how drafting works between mothers and calves and indicates that drafting is really important to the mother-calf bond," says Edwards, from the National Marine Fisheries Service. What is now open to question is the extent to which drafting operates at the pace created in the tuna-fishing chase, when adult dolphins move by a combination of bursts of speed, leaps out of the water and brief periods of coasting. "Reviewing the literature shows that in a chase situation the mothers will probably just run away and 'assume' that the babies can just draft along side," Edwards comments.

Edwards would also like to refine the model to specifically address the limits to drafting, and compare these limits to the expected conditions during purse-seine tuna-fishing operations. This will help to determine whether fishery-related mother-calf separations are a likely source of unobserved dolphin mortality in the eastern tropical Pacific Ocean.

So, dolphin researchers believe Weihs' paper to be a good step forward, but as with all good research it generates a new layer of questions. "Weihs has given us the mechanical energetics of the situation. What we now need is an understanding of how this translates into metabolic demand," says Fish. "The model also needs to be developed to take in all the movements of the dolphin." Doing that will move the mathematics closer to the mammal, and may ultimately help to reverse the tuna-fishing-related decline in dolphin numbers.

## Figures and Tables


author: Gerard D Wright
date: 2010
institute: 1Michael G DeGroote Institute for Infectious Disease Research and the Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main St W., Hamilton, Canada L8N 3Z5
title: Q&A: Antibiotic resistance: where does it come from and what can we do about it?

# Is antibiotic resistance inevitable?

Yes. Historically, the discovery of the sulfa drugs in the 1930 s and the subsequent development of penicillin during World War II ushered in a new era in the treatment of infectious diseases. Infections that were common causes of death and disease in the pre-antibiotic era - rheumatic fever, syphilis, cellulitis and bacterial pneumonia - became treatable, and over the next 20 years most of the classes of antibiotics that find clinical use today were discovered and changed medicine in a profound way. The availability of antibiotics enabled revolutionary medical interventions such as cancer chemotherapy, organ transplants and essentially all major invasive surgeries from joint replacements to coronary bypass. Antibiotics, though, are unique among drugs in that their use precipitates their obsolescence. Paradoxically, these cures select for organisms that can evade them, fueling an arms race between microbes, clinicians and drug discoverers.

# How do successful antibiotics work and what is the basis of resistance to them?

Antibiotics target essential bacterial physiology and biochemistry, causing microbial cell death or the cessation of growth. There are five major antibiotic targets: the bacterial cell wall, the cell membrane, protein synthesis, DNA and RNA synthesis, and folic acid (vitamin B9) metabolism (Figure 1). These bacterial targets are different or nonexistent in eukaryotic cells (including those of humans), which means that antibiotics are relatively nontoxic drugs. For example, the β-lactam antibiotics such as penicillins, cephalosporins and carbapenems block the synthesis of the bacterial cell wall. This structure is absent in higher organisms but is essential for bacterial survival. The bacterial ribosome is the target of the tetracycline, aminoglycoside, macrolide and other antibiotics, and is sufficiently different from the eukaryotic ribosome that cross-inhibition does not occur.

Resistance to antibiotics occurs through four general mechanisms: target modification; efflux; immunity and bypass; and enzyme-catalyzed destruction (Figure 1). Target modification can occur through mutation of the targets themselves - for example, the topoisomerases that are the target of the fluoroquinolone antibiotics - or by the production of enzymes that modify antibiotic targets, as, for example, in ribosomal methylation. Vancomycin resistance is a version of target modification where new biosynthetic machinery is engaged to alter cell-wall structure. Efflux occurs through a large family of protein pumps that eject antibiotics from inside the cell. In immunity, antibiotics or their targets are bound by proteins that prevent the antibiotic binding to its target. Arguably, the most specific and evolved mechanism of antibiotic resistance are enzymes that recognize antibiotics and modify them in such a way as to eliminate the functional characteristics that enable them to interact with their targets. For example, β-lactamases hydrolytically cleave the core β-lactam ring that is characteristic of the class and essential to antibiotic action.

# Has the problem of antibiotic resistance worsened over time?

Resistance to antibiotics was recorded even before the first clinical use of penicillin in the early 1940 s. In the intervening years, resistance to all classes of antibiotics has emerged, and there are no antibiotics for which resistance does not exist. There are two general strategies for resistance. One comprises mechanisms that transfer resistance vertically from a bacterium to its progeny. Examples are mutations in chromosomal genes that give rise to drug-insensitive products, such as the point mutations in the genes encoding DNA gyrase or topoisomerase IV that result in resistance to fluoroquinolone antibiotics such as ciprofloxacin. The second strategy includes the actions of genes that can be transmitted both vertically to progeny and horizontally to other bacteria, even those of different genera. These genes are located on mobile genetic elements such as plasmids, which can carry one or more resistance genes. Many of the β-lactamase genes that confer resistance to the penicillin, cephalosporin, penem and monobactam antibiotics are located on such elements, as is the glycopeptide-resistance gene cluster *vanHAX*, which provides resistance to vancomycin. The prevalence and mobility of resistance genes in previously sensitive pathogenic bacteria has now reached crisis levels in many cases because new antibiotics are no longer being developed at a rate that can keep pace with microbial evolution.

In the past two decades we have witnessed:

• the rise of so-called extended spectrum β-lactamases (ESBLs), which are mutants of enzymes that previously could only inactivate penicillins but now have gained activity against many cephalosporins;

• carbapenemases such as KPC and NDM-1 that inactivate all β-lactam antibiotics;

• plasmid-mediated (and thus horizontally disseminated) resistance to fluoroquinolone antibiotics;

• the spread of virulent MRSA (methicillin-resistant *Staphylococcus aureus*) in the community;

• the rise of multi-drug resistant *Neisseria gonorrhoea*;

• the emergence and global dissemination of multi-drug resistant *Acinetobacter baumannii*, *Pseudomonas aeruginosa*, *Klebsiella pneumoniae* and Enterobacteriaceae;

• the spread of extensively drug resistant *Mycobacterium tuberculosis*;

• the development of resistance to the two newest antibiotics to be approved for clinical use - daptomycin and linezolid.

Resistance is relentless and unavoidable as long as we use antibiotics.

# Where does resistance come from?

Antibiotic resistance is the evolutionary response to the strong selective pressure that results from exposure to these compounds. The horizontal dissemination of resistance genes into bacterial species and genera that are not themselves intrinsically resistant, as well as the maintenance of resistance mutations vertically through populations is likely to be the result of contemporary use of these drugs in the clinic and on the farm. Support for this hypothesis is the infrequency of antibiotic resistance in collections of pathogenic bacteria that pre-date the antibiotic era.

Nevertheless, antibiotic resistance is a natural phenomenon. It has been recognized for decades that the resistance mechanisms that have emerged in the clinic parallel those that are intrinsic to the bacteria that produce antibiotics. Recent studies of non-pathogenic soil bacteria have revealed that the majority of environmental bacteria tested are multi-drug resistant. This reflects the fact that these microbes live and have evolved in an environment where small bioactive molecules, some toxic, some benign, are plentiful and diverse. Bacteria have simply evolved to interact with them and control their biological effects. Pathogens, on the other hand, are often more virulent forms of our commensal bacteria and simply have not been exposed to the diversity and types of small molecules found in the environment; as a result, they have not required the gamut of resistance genes found in some environmental bacteria.

Furthermore, the genes and proteins responsible for resistance in environmental bacteria are homologous to those found circulating in pathogens, strongly suggesting contemporary horizontal gene transfer. Opportunistic pathogens with environmental reservoirs - for example, *P. aeruginosa* and *A. baumannii* - are highly drug resistant and have a remarkable capacity to acquire new resistance genes. The environment is therefore a large reservoir of potential resistance genes: the environmental 'resistome'.

Given the vast numbers of bacteria on the planet and the massive selection pressure provided by antibiotics, the movement of antibiotic-resistance elements from benign, but resistant, microbes into previously susceptible pathogens is simply a matter of time and opportunity.

# Can anything be done to slow down the emergence of resistance?

Antibiotics themselves are the source of the evolutionary pressure that eventually renders them obsolete. Limiting exposure of microbes to antibiotics therefore makes good sense to reduce the opportunity for the selection and dissemination of resistance. The inappropriate use of antibiotics by clinicians and the agricultural community needs to be curtailed. Over the past several years, the medical community in particular has made concrete efforts to curb the improper use of antibiotics. The European Union has taken the lead in limiting the non-therapeutic use of antibiotics in food animals. Robust surveillance networks that span the clinic and the farm need to be supported in order to monitor the impact of resistance and the emergence of new threats in real time. In North America, efforts such as the Strategies to Address Antimicrobial Resistance Act seek to diminish antibiotic use in agriculture and improve surveillance. Furthermore, there have been several successful campaigns to educate the public on the importance of antibiotics and the proper use of these drugs. While none of these efforts is perfect, there is much to be celebrated and encouraged.

These measures all serve to reduce antibiotic use and, as a result, delay the emergence of resistance. Furthermore, by decreasing selection pressure, the opportunity for the rise of particularly clinically challenging or virulent organisms should be lessened. All strategies that reduce the incorrect use of antibiotics are welcome, but in the end new drugs will always be needed because of the inevitability of resistance.

Unfortunately, in the developing world, access to antibiotics is frequently not regulated and their use in agriculture is often rampant. These facts make antibiotic stewardship especially challenging. In an era of rapid intercontinental travel, pathogens are no longer geographically contained and can move from country to country with ease. The recent examples of transcontinental spread of the severe acute respiratory syndrome (SARS) virus from Guangdong province in China to Hong Kong and then Canada in 2003, and the NDM-1 carbapenemase, which inactivates all β-lactam antibiotics and appears to have originated in the Indian subcontinent but is now found in North America, the UK and Europe, make the point.

# What about new antibiotics?

The growing problem of resistance fuels a continuous need for new antibiotic drugs. The enterobacteria that produce carbapenemase are just one example of antibiotic-resistant enterobacteria. Other Gram-negative pathogens resistant to virtually all antibiotics include multi-drug resistant *A. baumannii* and *P. aeruginosa*. The expanding problem of MRSA, and the global challenge of extensively drug-resistant *M. tuberculosis* (also called extreme drug-resistant *M. tuberculosis*), require new therapies.

There are some promising new candidates on the horizon, especially for the treatment of infections caused by Gram-positive pathogens such as MRSA and enterococci. As already mentioned, two new drugs active against this microbial spectrum - daptomycin and linezolid - have been introduced in the past decade. Tigecycline, a third-generation semi-synthetic tetracycline antibiotic approved in 2005, also has activity against MRSA. The semi-synthetic glycopeptide antibiotic telavancin recently received approval in the United States and the fifth-generation cephalosporin ceftobiprole is available in some European countries and Canada. However, there are few candidates in late-stage clinical trials suitable for the problem of Gram-negative pathogens. Here, often the choice of last resort is colistin, an antibiotic discovered more than 50 years ago and seldom used in the past because of adverse affects, including kidney toxicity; however, it is now increasingly used.

# Why are there so few new drugs?

There are a number of reasons, some economic, for the paucity of new antibiotics. They include challenging and shifting processes of government regulatory approval that add to the risk for the pharmaceutical industry. Furthermore, considerations of return on investment favor drugs for chronic diseases, which are taken by patients over long periods of time, often decades. In contrast, antibiotics cure disease and are taken for short periods of time.

Other reasons for the decline in antibiotic discovery and development are scientific. The first wave of antibiotics discovered five decades ago have been termed the 'low hanging fruit'. Despite the discovery of numerous compounds with antibiotic properties in the years since, few have had the requisite properties to become drugs. Most antibiotics are natural products or their derivatives that have been isolated from soil bacteria. Some researchers have suggested that this source might now be exhausted.

Furthermore, the promise of the genomic era and the reality of hundreds of available bacterial genomes have so far failed to deliver the hoped-for new molecular targets for antibiotics. Other new technologies, such as high-throughput screening of libraries of synthetic molecules, have not resulted in new drugs, although this may reflect a poor choice of chemical classes in the screens, emphasizing molecules more active in human biology than as antibiotics. Test compounds were often skewed in favor of small lipophilic molecules with physical properties meeting the criteria of Lipinski's Rule of 5. However, though helpful in assessing the prospect of a compound to be an orally active drug for human disease, this strategy has been shown to fail when searching for antibiotics.

# So what would be suitable chemical matter for leads?

Well, natural chemicals have significant advantages. Although the first antibiotics introduced into the clinic were the synthetic sulfonamides, the majority of antibiotics in current clinical use are bacterially produced natural products or their derivatives; only a few are completely synthetic in origin. The reasons for this in part reflect the history of antibiotic discovery post-penicillin, and the relative ease of discovery of suitable molecules through screening the products of soil microbes compared with libraries of synthetic compounds. Many of these 'natural' antibiotics have desirable drug-like qualities, such as good bioavailability, the ability to cross the cell membrane (and outer membrane in the case of compounds with Gram-negative activity) and the ability to evade efflux systems, and chemical structures that favor binding to vital cellular targets, supporting the idea that natural products encompass privileged structures in antibiotic drug discovery. However, the increasing difficulty of identifying new chemical compounds with equally suitable drug-like characteristics from natural sources has caused natural-product-based screening programs to fall out of favor in many pharmaceutical firms over the past few decades.

Instead, the ability of parallel synthesis methods to generate hundreds of thousands of synthetic molecules suitable for modern high-throughput screening has shifted the focus in favor of synthetic molecules in commercial antibacterial drug discovery. The advantages of synthetic compounds are not insignificant: pure lead molecules can easily be produced in quantity and quality suitable for clinical trials, and are relatively easily modified to improve target affinity. However, after two to three decades of emphasis on such molecules and millions of dollars spent on high-throughput *in vitro* and cell-based screens, no new synthetic antibiotics have emerged. Linezolid, the one synthetic antibiotic to be brought to market in the past decade, was discovered using traditional medicinal chemistry in a research program with a plant-disease focus in the early 1980 s.

# So does that mean natural products are best after all?

They do have great advantages, although a direct comparison of the success and failure of synthetic as against natural product libraries is unfair. Microbial natural products have evolved over millennia to interact with biological molecules, whereas the synthetic chemical libraries used in antibiotic drug-discovery screens were generally developed with a focus on eukaryotic drug-discovery campaigns, as noted earlier. Efforts to develop physical-property rules for antibiotics and to incorporate natural-product-like chemical complexity in libraries of synthetic chemicals will no doubt improve success in identifying new synthetic antibiotic leads.

Ironically, at the same time that the pharmaceutical industry was abandoning natural-product libraries, university researchers were making remarkable advances in understanding the molecular details of natural-product biosynthesis by bacteria. Many bacteria, especially the actinomycete group of common environmental bacteria, are prodigious producers of natural products. These are termed secondary metabolites to contrast with molecules of primary metabolism, such as carbohydrates, amino acids and so on. Secondary metabolites range in molecular weight from around 100 daltons (Da) to up to 5,000 Da and they have diverse biological activities, including induction of cell death (antibiotics such as tetracycline, vancomycin and daptomycin, and anticancer agents such as adriamycin), iron sequestration (for example, enterobactin), facilitation of cell-cell communication (γ-butyrolactones), protection from oxidizing agents (phenazines), and a host of others.

The bacterial natural products that are most important as antibiotics include polyketides, such as the macrolides and tetracyclines, and non-ribosomal peptides - that is, peptides that are not synthesized on ribosomes - which include β-lactams and glycopeptides such as vancomycin. These are produced in the cell in assembly-line fashion on large dedicated enzyme platforms called, respectively, polyketide synthases and non-ribosomal peptide synthetases. Following assembly the compounds are then 'decorated' by a series of modifying enzymes, such as glycosyltransferases. The end result is a molecule of often complex structure, with multiple chiral centers and functional groups such as sugars, halogens, sulfates, acyl groups and others.

In general, bacterial genes that encode the production of natural products are clustered together in the genome, greatly facilitating analysis and prediction of biosynthetic pathways and structures. Indeed, several software packages (for example, NP. searcher) have been developed based on rules-based understanding of natural-product biosynthesis. The availability of cheap, rapid genome sequencing means that time-consuming construction and screening of gene libraries for natural-product clusters can now be bypassed. Genome sequencing has also revealed a hitherto unrealized richness in the quantity and variability of natural-product biosynthetic clusters. Sequenced genomes of bacteria of the actinomycetes class reveal 20 to 30 biosynthetic clusters in each organism. Furthermore, natural-product producing bacteria from non-soil environments are being investigated and these have already resulted in new chemical matter, suggesting that there is a fantastic wealth of untapped chemical diversity waiting to be discovered. Perhaps some of this diversity will include new antibiotic chemical scaffolds.

We are in a remarkably productive time for natural-product research that is serving to reinvigorate interest in this sector. At the same time, the application of synthetic biology approaches to this field could serve to improve issues of yield and expand chemical diversity.

# Are there alternatives to new antibiotics?

Yes. First, existing discarded antibiotics can be re-examined. The development of daptomycin is instructive. Daptomycin was discovered by the antibiotic group at Eli Lilly in the 1980 s, but was not fully developed because of toxicity concerns. The antibiotic was obtained by researchers at Cubist in 1997 and by altering the dosing, this group was able to bring the antibiotic to market in 2003, since when it has proved highly successful in treating infections caused by Gram-positive pathogens. Certainly, there are other 'old' antibiotics discovered by the pharmaceutical industry but not developed at the time that could be resurrected as leads for new drugs.

A second option is the combination of antibiotics with each other and with other drugs to improve efficacy. Infectious-disease physicians often combine antibiotics in an effort to achieve synergy, and this well-established practice has resulted in formulated drug combinations, such as co-trimoxazole (trimethoprim and sulfamethoxazole). Combination of antibiotics with non-antibiotics deserves investigation as well. Several natural products have been discovered by Satoshi Omura's group that potentiate the activities of antibiotics such as imipenem in *S. aureus* by unknown mechanisms.

Other antibiotic adjuvants are inhibitors of resistance mechanisms. The tremendous commercial and clinical success of Augmentin (ampicillin together with the β-lactamase inhibitor clavulanic acid) and other similar combinations speaks to the power of such combinations. Our growing understanding of the mechanisms of resistance should fuel such approaches. Inhibitors of efflux pumps, for example, have been discovered, and though challenging to implement in organisms with multiple redundant systems, are worthy of continued investigation.

Finally, other strategies orthogonal to antibiotics must be on the table. We should never forget vaccines as proven and outstanding protective agents against infectious diseases. Bacterial viruses (bacteriophages) were used extensively to treat bacterial infections in the former Soviet Union and could find new application in the face of outbreaks of multi-drug resistant bacteria, especially in settings such as hospital infections. The use of enhancers of innate immunity, such as cationic antimicrobial peptides, is also an approach worth investigating.

# What is the outlook for new drugs and further resistance?

We need antibiotics to maintain our current standard of health care. As already stated, resistance is a natural evolutionary phenomenon that cannot be stopped. Through judicious use of current drugs and the development of new ones, the pace of resistance development can be controlled without impairing our ability to control disease. The need for new drugs is, however, acute. Antimicrobial stewardship alone cannot fulfill our requirement for new antibiotics.

We are in a remarkably exciting time for fundamental research in antibiotics. The rapidity of genome sequencing, the maturing of our knowledge of natural product biosynthesis, a growing understanding of the physical properties of ideal antibiotics, the development of new strategies to develop synthetic compounds with improved antibiotic properties, and the possibilities of synthetic biology combine to suggest that we are entering a highly productive period of antibiotic discovery. The challenges of moving these advances into the clinic fast enough to keep pace with resistance are significant, but with concerted effort between scientists, funders, industry, regulators and clinicians, I believe they can be overcome.

# Where can I go for more information?

## Articles

Arias CA, Murray BE: **Antibiotic-resistant bugs in the 21st century - a clinical super-challenge**. *N Engl J Med* 2009, **360:**439-443.

Baquero F, Alvarez-Ortega C, Martinez JL: **Ecology and evolution of antibiotic resistance**. *Env Microbiol Rep* 2009, **1:**469-476.

Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice LB, Scheld M, Spellberg B, Bartlett J: **Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America**. *Clin Infect Dis* 2009, **48:**1-12.

Fenical W, Jensen PR: **Developing a new resource for drug discovery: marine actinomycete bacteria**. *Nat Chem Biol* 2006, **2:**666-673.

Fischbach MA, Walsh CT: **Antibiotics for emerging pathogens**. *Science* 2009, **325:**1089-1093.

Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, Chaudhary U, Doumith M, Giske CG, Irfan S, Krishnan P, Kumar AV, Maharjan S, Mushtaq S, Noorie T, Paterson DL, Pearson A, Perry C, Pike R, Rao B, Ray U, Sarma JB, Sharma M, Sheridan E, Thirunarayan MA, Turton J, Upadhyay S, Warner M, Welfare W, Livermore DM, Woodford N: **Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study**. *Lancet Infect Dis* 2010, **10:**597-602.

Livermore DM: **Has the era of untreatable infections arived?** *J Antimicrob Chemother* 2009, **64 Suppl 1:**i29-i36.

Shahid M, Sobia F, Singh A, Malik A, Khan HM, Jonas D, Hawkey PM: **Beta-lactams and beta-lactamase-inhibitors in current- or potential-clinical practice: a comprehensive update**. *Crit Rev Microbiol* 2009, **35:**81-108.

Wright GD: **The antibiotic resistome: the nexus of chemical and genetic diversity**. *Nat Rev Microbiol* 2007, **5:**175-186.

Yamazaki H, Nonaka K, Masuma R, Omura S, Tomoda H: **Xanthoradones, new potentiators of imipenem activity against methicillin-resistant Staphylococcus aureus, produced by *Penicillium radicum* FKI-3765-2: I. Taxonomy, fermentation, isolation and biological properties**. *J Antibiot (Tokyo)* 2009, **62:**431-434.

## Web links

**Infectious Diseases Society of America (IDSA)** <http://www.idsociety.org/>

## Acknowledgements

Research in the author's lab on antibiotic resistance is supported by a Canada Research Chair, the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council.


author: Margitta Worm
date: 2011
institute: 1Department of Dermatology and Allergy, Allergy Center Charité, CCM, Charité - Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany
title: Q&A: Food additive intolerance

# Q&A: Food additive intolerance

## 1) What is food additive intolerance and can you tell us what the most common symptoms are?

Food additive intolerance is a non-IgE mediated food hypersensitivity. The hypersensitivity is induced by the food additives via a **direct** mast cell activation. Although the exact pathophysiology is unknown, various clinical signs are characteristic of food additive intolerance. These include cutaneous symtoms like redness of the skin, urticaria and angioedema as well as other organ related symptoms, such as dyspnea, hypotension or dizziness.

## 2) What are the differences between intolerance and allergy?

Both, IgE-mediated allergy, but also food additive intolerance are mast cell dependent reactions. The release of mast cell mediators like histamine, leukotrienes and others leads to the onset of the above mentioned clinical symptoms, which cannot be distinguished concerning the underlying mechanisms. In the case of an allergic reaction, crosslinking of membrane bound IgE via an allergen is inducing mast cell degranulation. In food additive intolerance a direct action of the additive on the mast cells is proposed, however the exact mechanisms are not known.

## 3) What are the most common food additives that cause intolerance?

The most common food additives to which patients are intolerant are sulfite, sodium benzoate and food colorings. In addition, histamine is often accused of inducing intolerance reactions, however its exact role as an intolerance reaction as such requires more clarification.

## 4) What is the prevalence of food intolerance?

It is estimated that 0.1 - 1.5% of the population may suffer from food additive tolerance. So far the data suggest that intolerance reactions occur more frequently in older patients rather than in young children Further, it is known that drug intolerance towards acetylsalicylic acid (ASA) occurs more frequently in asthmatic patients. However, whether this is also true in respect to the prevalence of food intolerance in asthmatics remains to be determined.

## 5) Is there an age dependent increase in allergy risk and what are the reasons for this?

The risk of developing an allergy to my knowledge does not solely depend on age but rather depends on the allergen and the specific exposure situation of an individual. The lifetime risk for a food allergy probably does decrease rather than increase over time. However, in the case of food additive intolerance, this decrease of the risk over time might not be true. A possible explanation for this might be a change of the gastrointestinal barrier function. In addition the presence of additional cofactors, which can trigger such reactions (intake of drugs like ACE-inhibitors, physical activity, in-take of alcohol), makes the onset of food additive intolerance in later life more likely.

## 6) Are there any co-morbidities that increase the risk of being intolerant to food additives?

As mentioned above, probably patients with moderate to severe asthma are at a higher risk of being of intolerant to food additives. In addition, it is well known that patients who suffer from mastocytosis, a genetic disease where mast cells occur in increased numbers in the skin and other organs have an increased risk to develop systemic-reactions to food additives

## 7) What are the current diagnostic and management strategies for food additive intolerance?

To date, the optimal management strategies for food additive intolerance include the avoidance of an increased intake of food additives in general, in particular in large amounts. For example, a meal with ripened cheese, wine and a colored dessert should be avoided. If a patient has skin and gastrointestinal symptoms, a prophylactic intake of antihistamines might be useful. Diagnostic procedures involve an elimination diet (3 - 4 weeks) followed by double blind-placebo-controlled-food challenge (DBPCFC) tests. Only if the double blind-placebo-controlled-food challenge is positive the diagnosis can be proven and dietary recommendations be made. Previous data of patients suffering from chronic urticaria has indicated a change in diet can facilitate gastrointestinal barrier recovery, which enables the patient to include certain food items step by step again over time again.

## 8) Are there any difficulties in the diagnosis of food additive intolerance?

The major difficulties in the diagnosis of food additive intolerance are that the history of symptoms made by the patients might not be clear. In such cases, a symptom diary might be helpful. It is important to note that *in vivo* tests, such as the skin prick-test, and *in vitro tests* such as determination of specific IgE cannot be used to make the diagnosis. Moreover, other methods such as the cellular activation test (CAST) measuring histamine release and/or the leukotriene pathway production can not be recommended to confirm the diagnosis. Therefore, research in this area is urgently required. This would help to improve the diagnostic methods of food additive intolerance, identify patients at risk and would support the development of new therapeutic strategies. The lack of knowledge in this field is e.g. related to the fact that food additive intolerance cannot be studied well in vitro as mast cell reactivity is different if studied in vitro versus in vivo.

## 9) What does the future hold for the diagnosis and management of food additive allergies?

I am sure that in the future we will be able to come up with novel diagnostic approaches. These might be based on cellular test systems and/or a metabolomic analysis from patients' serum. In particular, mediators and their receptors from the leukotriene pathway should be considered in more detail as data from previous studies suggest that genetic polymorphisms of leukotriene receptors may be influential, as well as deviations within the leukotriene profile might be relevant. In terms of medical strategies, the exact role of the gastrointestinal barrier will need more clarification. If methods can be developed to increase the barrier function of the gut these might also offer novel approaches for food intolerant patients because less absorption or enhanced local degradation of the food additive in the gut would decrease the systemic burden.

## 10) Where can I find out more?

Soost S, Leynaert B, Almqvist C, Edenharter G, Zuberbier T, Worm M. Risk factors of adverse reactions to food in German adults. Clin Exp Allergy. 2009 Jul;39(7):1036-44. Epub 2009 Mar 3

Roehr CC, Edenharter G, Reimann S, Ehlers I, Worm M, Zuberbier T, Niggemann B. Food allergy and non-allergic food hypersensitivity in children and adolescents. Clin Exp Allergy. 2004 Oct;34(10):1534-41

Zuberbier T, Edenharter G, Worm M, Ehlers I, Reimann S, Hantke T, Roehr CC, Bergmann KE, Niggemann B. Prevalence of adverse reactions to food in Germany - a population study. Allergy. 2004 Mar;59(3):338-45

Fiedler EM, Zuberbier T, Worm M. A combination of wheat flour, ethanol and food additives inducing FDEIA. Allergy. 2002 Nov;57(11):1090-1. No abstract available

Ehlers I, Hipler UC, Zuberbier T, Worm M. Ethanol as a cause of hypersensitivity reactions to alcoholic beverages. Clin Exp Allergy. 2002 Aug;32(8):1231-5

Worm M, Vieth W, Ehlers I, Sterry W, Zuberbier T. Increased leukotriene production by food additives in patients with atopic dermatitis and proven food intolerance. Clin Exp Allergy. 2001 Feb;31(2):265-73.

Worm M, Ehlers I, Sterry W, Zuberbier T. Clinical relevance of food additives in adult patients with atopic dermatitis. Clin Exp Allergy. 2000 Mar;30(3):407-14

Brockow K, Jofer C, Behrendt H, Ring J. Anaphylaxis in patients with mastocytosis: a study on history, clinical features and risk factors in 120 patients. Allergy. 2008 Feb;63(2):226-32

Reese I, Zuberbier T, Bunselmeyer B, Erdmann S, Henzgen M, Fuchs T, Jäger L, Kleine-Tebbe J, Lepp U, Niggemann B, Raithel M, Saloga J, Vieths S, Werfel T. Diagnostic approach for suspected pseudoallergic reaction to food ingredients. J Dtsch Dermatol Ges. 2009 Jan;7(1):70-7. Epub 2008 Nov 24. Review

Buhner S, Reese I, Kuehl F, Lochs H, Zuberbier T. Pseudoallergic reactions in chronic urticaria are associ-ated with altered gastroduodenal permeability. Allergy. 2004 Oct;59(10):1118-23