Subjects

The subjects were recruited through advertisements in the popular media and presentations at preschool programs. Sugar-sensitive children were identified on the basis of reports by their parents.

Two cohorts of children were studied concurrently. One group consisted of 23 children of primary-school age (6 to 10 years) reported by their parents to respond adversely to sugar, and the other group consisted of 25 normal preschool-age children (3 to 5 years of age).

Design

Table 1. Table 1. Diet Sequences for Two Subjects in a Latin-Square Design for Experimental Diets with a Random Distribution of Sham Diets.

The study protocol was approved by the University of Iowa Committee on Research Involving Human Subjects. The subjects and their families were placed on a different diet for each of three consecutive three-week periods. One of the three diets was high in sucrose with no artificial sweeteners, another was low in sucrose and contained aspartame, and the third was low in sucrose and contained saccharin (the placebo). All diets were essentially free of additives, artificial food coloring, and preservatives. Two of the diet sequences are shown in Table 1.

The children, their families, and the research staff were kept unaware of the sequence of the diets. This blinding was reinforced by visible changes made weekly in the diets (sham diets), but the sweetener was changed only every third week. Families were informed that the diets would change weekly and were given a list of the dietary components that were either varied or controlled. The parents were asked to identify the experimental variables each week, if they could.

Provision of Diets

Immediately before the beginning of the study, a dietitian supervised the removal of food from the children's home. During the nine-week study period, all foods were provided for the subject and his or her immediate family. Family members were allowed items not included in the diet when they were out of the home and away from the subject, and coffee and alcohol were allowed to remain in the home as long as they were not consumed by the children. The food was delivered in a van equipped to serve as a mobile testing laboratory. All food was removed from the home at the end of each week, and a new supply delivered. In addition, parents kept records of all food consumed by the subjects and were encouraged to report any deviations from the specified diet.

During each three-week diet period, foods were sweetened with sucrose, aspartame, or saccharin, depending on which diet was assigned for that period. Care was taken to keep the appearance of the sweetened products identical, regardless of the sweetener used. Sweetened foods included pure fruit juice, fruit, cereals, pudding, flavored yogurt, cookies, fruit toppings, and bottled carbonated soft drinks. The soft drinks were supplied by three national bottlers in unmarked but coded bottles; unsweetened fruit juice and unsweetened cereal were provided by two national distributors. Small amounts of saccharin were used to sweeten items, such as condiments, that were consumed in small amounts by the subjects during all nine weeks. The use of various food components popularly believed to influence behavior was kept to a minimum. These included artificial colors, artificial flavors, additives, monosodium l-glutamate, chocolate, and caffeine. Shortenings and oils used in the diets did not contain butylated hydroxyanisole or butylated hydroxytoluene, and the use of frozen meats and baked products treated with these antioxidants was kept to a minimum. The three sham diets consisted of abundant quantities of red and orange foods (diet A); beef and pork, with only raw fruits and vegetables (diet B); and chicken and fish, with only cooked fruits and vegetables (diet C) (Table 1).

Dietary Intake

Dietary intake was documented in diaries by the parents and reviewed weekly with the dietitian. Parents were taught how to estimate the amounts of food consumed, and each week they received seven diary sheets listing the daily menus. The study design imposed no restrictions on the quantity of each food consumed, and parents were asked not to restrict access to any food.

Compliance

To determine dietary compliance, 1 mg of ascorbic acid per milligram of aspartame was added to foods sweetened with aspartame, and 1 mg of riboflavin per 5 g of sucrose was added to foods sweetened with sucrose. These concentrations provide at least 10 times the recommended dietary allowances,9 with the amount excreted roughly proportional to the amount of sweetener ingested. Urine samples were obtained weekly and tested for ascorbate, riboflavin, and creatinine10-12.

Behavioral and Cognitive Measures

Table 2. Table 2. Cognitive and Behavioral Measures Administered Weekly.

The children were initially evaluated by means of a structured psychiatric interview with the parent (Diagnostic Interview Schedule for Children -- Parent Version),13 the Wechsler Intelligence Scale for Children -- Revised14 or the Wechsler Preschool and Primary Scale of Intelligence,15 and the Wide Range Achievement Test -- Revised16 (for school-age children only). At base line and during each of the nine weeks of the study, the subjects were evaluated with behavioral and cognitive measures hypothesized to be sensitive to the effects of sweetener (Table 2). During base-line testing, children were oriented to the tests, and appropriate levels of difficulty were determined for the learning and academic tasks. This served to minimize the effects of practice during the study period. Motor activity was assessed with a solid-state device for measuring motion28. Five-second time sampling was used to assess behavior and activity levels during the performance of a writing or drawing task29. Several tasks (e.g., card sorting and academic tests) were too difficult to be administered to the preschool children. Children rated their mood and physical state on a visual-analogue scale20 adapted from a self-report measure used to assess the effects of stimulants in previous studies30. Measures were administered in the mobile laboratory each week on the same day of the week and at the same time of day. In addition, structured ratings of specific types of behavior were completed by the children's parents, the children's teachers, and research assistants (Table 2). For the preschool-age children, the teacher's rating was completed by a preschool teacher or a care giver other than the mother.

Biochemical Tests

Base-line biochemical tests included a fasting sucrose-tolerance test and fasting plasma amino acid analyses. Sucrose tolerance was calculated by determining blood glucose concentrations in samples drawn at 0, 0.5, 1, 2, 3, and 4.5 hours after a sucrose drink (1.75 g per kilogram of body weight). On the third, sixth, and ninth weeks of the study, postprandial blood samples for plasma glucose and amino acid analyses were drawn 2 to 3 hours after a meal and 30 to 60 minutes after the subject had drunk 250 ml (8 oz) of an uncarbonated beverage (providing 170 mg of aspartame, 30 g of sucrose, or 40 mg of saccharin). Plasma glucose concentrations were assessed by the glucose-6-phosphate dehydrogenase method. Plasma amino acid levels were determined with automated amino acid analyzers (Beckman 121 MB; Beckman Instruments, Palo Alto, Calif.),31 and these calculations included the molar ratio of the plasma phenylalanine concentration to the sum of the concentrations of the other large neutral amino acids32 sharing its transport to the brain.

Statistical Analysis

The analysis was designed to evaluate a large number of possible effects. At the same time, it was important that significance levels not be set so conservatively that we would incorrectly accept the null hypothesis despite clinically important differences; for this reason, Bonferroni corrections were not applied. Similarly, multivariate analyses were not used because of concern about a reduction in the degrees of freedom and difficulty in handling selective missing data in such analyses. Separate repeated-measures analyses of variance were carried out for each dependent variable, and individual comparisons among the three treatments were made with Tukey's test, each at the 0.05 level of significance. Although this approach increased the chance of a type I error, it maximized our ability to detect any differences attributable to the sweeteners. The use of a counterbalanced Latin square design33 eliminated the possibility that practice or the sequence of the diets would account for differences in the cognitive or behavioral variables.

To increase the reliability of the cognitive and behavioral assessments, the three scores obtained for each dietary period were averaged, and these mean scores were compared by a repeated-measures analysis of variance. To detect possible cumulative effects, the results from the final week of each period were also compared. In addition, analyses of variance were performed to determine whether the sham diets had any influence on the cognitive and behavioral variables. Dietary intake was calculated for each treatment period and for each sham diet during that period. The intake of macronutrients, sweeteners, and vitamin markers was tabulated, and differences were evaluated with an analysis of variance.

To identify individual subjects who may have responded adversely to sugar or aspartame, the weekly scores for each of nine core neurobehavioral measures were ranked and examined to determine whether the poorest scores were clustered during a particular dietary period. This approach was considered preferable to setting a fixed cutoff point for differences (e.g., a 25 percent change) because of the wide variation in raw scores among the variables studied and in score levels at different ages.