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NNNS: Adverse Effects (2011)

Citation:
 
Study Design:
Class:
- Click here for explanation of classification scheme.
Quality Rating:
Research Purpose:
To determine whether aspartame can disrupt cognitive, neurophysiologic or behavioral functioning in normal individuals.
Inclusion Criteria:
  • Did not have medical, neurological or psychiatric disorders, phenylketonuria or learning disabilities
  • Pre-tested and had to have at least average (above the 50th percentile) verbal and non-verbal intelligence, verbal attention and memory.
Exclusion Criteria:
  • Pregnant or lactating women
  • Taking oral contraceptives
  • Taking psychotropics or with a history of drug or alcohol abuse.
Description of Study Protocol:

Recruitment

  • The study was conducted at MIT CRC

  • Electroencephalograms were done at Beth Israel Deaconess Medical Center in Boston

  • Protocol was approved by the human research committees of both institutions

  • Subjects were paid for participation

  • Subjects were recruited by advertisement.

 
Design

  • Pre-tested to ensure that they were of at least average (above the 50th percentile) verbal and non-verbal intelligence, verbal attention and memory, before they proceeded to familiarization with a computer-administered task (think fast; 19) dependent measure

  • Potential subjects underwent electrocardiogram, an EEG, routine hematologic tests, urinalysis, blood chemistry tests, urine and plasma toxin screens and routine physical and neurologic examinations.

Treatments
  • Asparatame
  • Sucrose
  • Placebo
  • Randomized double-blind three-way crossover design: Subjects were assigned to either high (HIAP: 45mg per kg of body weight -1 D-1) or low (LOAP: 15mg per kg of body weight -1 D-1) doses of aspartame
  • HIAP approached the FDA’s acceptable daily intake for aspartame (50mg per kg of body weight -1 D-1)
  • Equal numbers of men and women were randomly assigned to the HIAP and LOAP groups
  • Sucrose treatment (90g per day) was the same for all subjects
  • Sucrose delivery in beverage: A lightly carbonated lemon-lime flavored soda
  • Placebo sodas were unsweetened and capsules contained 300mg microcrystalline cellulose and 0.9mg silicon dioxide
  • Sodas were identical in appearance, except for being labeled "Treatment One," "Treatment Two" or "Treatment Three." The number of capsules varied by aspartame dose and body weight, but identical numbers of capsules were given each treatment period.
  • Subjects were not permitted to consume dietary aspartame during the study, but dietary sucrose was restricted on test days. Subjects maintained their usual diets, except on test days, when meals were provided and they were asked not to snack or consume additional sucrose.
  • Compliance was verified by plasma amino acids and glucose analysis.
Procedures
  • Four consecutive months with three outpatient visits per treatment period: Two for testing and one for monitoring before the next treatment
  • Baseline: First month, during which subjects were asked not to consume aspartame and were not given any treatment
  • During Months Two, Three and Four, subjects were provided with sodas and capsules, which they took three times daily (at 10:00 a.m., 3:00 p.m. and 8:00 p.m.) for 20 days. Order was randomized so that each of the six orders for the three treatments was assigned eight times.
  • Neuropsychologic testing: Done on Days 10 and 20 of each treatment period. Female subjects' testing was done on the 10th and 20th days of their menstrual cycles to minimize any ovulatory or late-luteal influences. Acute effects (Day 10) were assessed 1.5 hours after capsules and sodas were ingested; chronic effects (Day 20) by testing before any morning treatments were consumed.
Schedule of Treatments and Procedures on the Acute (Day 10) and Chronic (Day 20) Testing Days1
Time Blood Draw Toxin Screen Neurologic Examination Physical Examination Prepared Breakfast Treatment Substance Taken Prepared Lunch Profile of Mood States Neuropsychologic Testing
8:00 a.m.
 XO O XO XO          
8:15 a.m.         XO     O  
8:30 a.m.                 O
9:55 a.m. O                
10:00 a.m.           XO X    
2:45 p.m.               X  
2:55 p.m. X                
3:00 p.m.           X      
4:20 p.m.               X   
4:25 p.m. X   X            
4:30 p.m.                 X 
5:30 p.m. X                
 
1: X=Day 10, O=Day 20

Meals were standardized on test days and breakfast contained 11g protein, six grams fat and 75g carbohydrate. Lunch provided 34g protein, 16g fat and 107g carbohydrate for men and 26g protein, nine grams fat and 84g carbohydrates for women.

Blinding Used

  • Results of randomization were only known to the clinical pharmacy that prepared the treatment supplies and to an independent statistician. Sodas and capsules were administered for all treatments to maintain blinding.
  • Subjects were told that the treatment might be contained in either the capsules or the sodas and that taste might not be correlated with treatment. The blinding was tested in a Latin-square design for the first 12 subjects who guessed their order of treatment. Independent statistical analysis showed that the blinding was effective.

Statistical Analysis

  • Significance set at P<0.05.
  • The neurocognitive tests, POMS factors, amino acids, Phe:LNAA values and all blood values, including glucose and insulin, were analyzed by a series of univariate multi-factor analyses of variance (ANOVAs). The adverse experiences and EEGs were analyzed with the exact form of McNemar’s test for correlated proportions.
Data Collection Summary:

Dependent

  • Physical: Included supine and standing blood pressure, weight, temperature and heart and respiratory rates. Brief physical and neurologic examinations with strength and reflex testing.
  • Laboratory: Blood was collected for glucose, insulin and amino acid tests; for toxin screens and for routine hematology (hemoglobin and hamatocrit and red blood cell, white blood cell, differential and platelet counts) and fasting blood chemistyry tests (total protein, albumin, calcium, inorganic phosphorus, cholesterol, glucose, urea nitrogen, uric acid, alkaline phosphatase, lactate dehydrogenase, total bilirubin, serum aspartate aminotransferase, alaine aminotransferase, sodium, potassium, chloride, carbon dioxide and creatinine). Samples were also obtained for urinalysis.
  • Amino acids, glucose, insulin concentration, toxin screens, urine screens for drugs of abuse and a serum screen for alcohol were done
  • Adverse experience: Evaluated at each visit. Time of onset, duration, intensity and frequency were recorded for any adverse physical, cognitive or behavioral symptom.
  • Neurophysiology: EEGs were performed with a Grass machine by using international 10-20 system montages for electrode placement. Recording was for 45 minutes, with a brief hyperventilation and photic stimulation and were read by board certified electroencephalographers, who were blinded to treatment and did not interact with the subjects.  
  • Neuropsychology: Cognitive measures were selected to screen functioning in the frontal and temporolimbic networks, which are responsible for many aspects of mood, memory and behavior.  
Table Three: Neuropsychologic Functions Tested, Measures Given and Order of Administration at Each Testing Session on the Acute (Day 10) and Chronic (Day 20) Testing Days

Function Test Administered
Verbal learning 20-word list, five trials, free recall
Verbal attention span Digit span, forward and backward
Spatial attention span Corsi block test, forward and backward
Short-term memory Free recall of word list
Verbal fluency Controlled oral word association
Response set alternation Trailmaking tests, Form A and Form B
Response set inhibition Stroop test, interference condition
Motor response set alternation Auditory reciprocal motor programs
Motor response set inhibition Auditory, go/no-go 
Overall cognitive efficiency Think fast
Long-term memory Free recall of word list
  • Mood: POMS ratings were for six scales (tension, anger, depression, vigor, fatigue and confusion). To assess substance-dependent effects on mood, subjects completed the POMS on Day 10, before their afternoon treatment and then later, before neuropsychologic testing. The POMS on Day 20 was completed in the morning before the treatment substance was consumed.
Description of Actual Data Sample:
  • Initial N: 76 undergraduate or graduate students were screened to obtain 48 (24 males and 24 females)
  • Attrition (final N): Three subjects discontinued (two men moved away and one woman required surgery). No drop-outs resulted from adverse experiences related to the treatment. Drop-outs were replaced to keep the statistical power.
  • Age: 18-35 years
  • Ethnicity: 23% were Asian, Eurasian or African-American and the remainder were white
  • Location: Boston.
Summary of Results:
  • Laboratory: Toxin screens and laboratory values confirmed that subjects were compliant with the protocol
  • Amino acid analyses: Showed significant differences during the aspartame treatment
  • Acute test days: Both aspartame groups had significant increases in Phe:LNAA, compared with their values when receiving placebo and sucrose (P<0.05).
Plasma Concentrations of Phenylalanine (Phe) and the Ratio of Phenylalanine to All Other Large Neutral Amino Acids (Phe:LNAA) on the Acute Testing Days (Day 10), Before and After Neuropsychologic Testing1
  Blood Sampling Time Blood Sampling Time Blood Sampling Time Blood Sampling Time
  4:25 p.m.
 4:25 p.m.
 5:30 p.m.
 5:30 p.m.
Group and Treatment Phe umol/L

Phe:LNAA
umol/L

 Phe umol/L

Phe:LNAA
umol/L
HIAP (N=24)    
Aspartame

78.5±12.22
(56.8-104.1)

0.138±0.0232
(0.101-0.183)

71.8±8.72
(53.0-87.0)

0.132±0.0162
(0.101-0.169)
Placebo  56.2±10.4
(42.0-83.6)		 0.097±0.010 (0.075-0.116)  52.8±10.6 (36.0-84.0)  0.095±0.012 (0.066-0.117) 
Sucrose  56.3±8.9 (41.4-77.9) 0.103±0.011 (0.084-0.129)  50.4±5.6 (40.9-60.6)  0.096±0.009 (0.079-0.110) 
LOAP (N=24)    
Aspartame

67.9±10.22
(56.8-106.5)

0.118±0.0222
(0.087-0.195)

60.7±10.62
(43.2-95.7)

0.114±0.0222
(0.072-0.196)
Placebo 61.0 ± 9.8 (42.8-86.5)

0.105 ± 0.018
(0.075-0.158) 

 57.2±9.4 (38.1-75.30)  0.104±0.015 (0.079-0.137) 
Sucrose 55.2±6.7 (43.2-71.3) 0.102±0.013 (0.080-0.128) 51.6±5.1 (38.9-62.7) 0.098±0.007 (0.085-0.113)

1: Mean±SD, range in parentheses
2: Significantly different from placebo and sucrose treatments, P<0.05 (planned comparison).

  • Neuropsychologic results, adverse experiences, amino acids, insulin and glucose values and electroencephalograms were compared by sex and by treatment. No significant differences were found for any dependent measure.
  • Large daily doses of aspartame had no effect on neuropsychologic, neurophysiologic or behavioral functioning in healthy young adults.
Author Conclusion:

The dose of aspartame taken by out HIAP group was nearly 20 times the 90th percentile average daily intake of aspartame and still did not result in adverse behavioral, neuropsychologic or neurophysiologic effects. Consequently, we conclude that aspartame is safe.

Funding Source:
Reviewer Comments:
Quality Criteria Checklist: Primary Research
Relevance Questions
  1. Would implementing the studied intervention or procedure (if found successful) result in improved outcomes for the patients/clients/population group? (Not Applicable for some epidemiological studies) Yes
  2. Did the authors study an outcome (dependent variable) or topic that the patients/clients/population group would care about? Yes
  3. Is the focus of the intervention or procedure (independent variable) or topic of study a common issue of concern to dieteticspractice? Yes
  4. Is the intervention or procedure feasible? (NA for some epidemiological studies) Yes
 
Validity Questions
1. Was the research question clearly stated? Yes
  1.1. Was (were) the specific intervention(s) or procedure(s) [independent variable(s)] identified? Yes
  1.2. Was (were) the outcome(s) [dependent variable(s)] clearly indicated? Yes
  1.3. Were the target population and setting specified? Yes
2. Was the selection of study subjects/patients free from bias? Yes
  2.1. Were inclusion/exclusion criteria specified (e.g., risk, point in disease progression, diagnostic or prognosis criteria), and with sufficient detail and without omitting criteria critical to the study? Yes
  2.2. Were criteria applied equally to all study groups? Yes
  2.3. Were health, demographics, and other characteristics of subjects described? Yes
  2.4. Were the subjects/patients a representative sample of the relevant population? Yes
3. Were study groups comparable? Yes
  3.1. Was the method of assigning subjects/patients to groups described and unbiased? (Method of randomization identified if RCT) Yes
  3.2. Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline? Yes
  3.3. Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.) Yes
  3.4. If cohort study or cross-sectional study, were groups comparable on important confounding factors and/or were preexisting differences accounted for by using appropriate adjustments in statistical analysis? N/A
  3.5. If case control study, were potential confounding factors comparable for cases and controls? (If case series or trial with subjects serving as own control, this criterion is not applicable.) N/A
  3.6. If diagnostic test, was there an independent blind comparison with an appropriate reference standard (e.g., "gold standard")? N/A
4. Was method of handling withdrawals described? Yes
  4.1. Were follow-up methods described and the same for all groups? Yes
  4.2. Was the number, characteristics of withdrawals (i.e., dropouts, lost to follow up, attrition rate) and/or response rate (cross-sectional studies) described for each group? (Follow up goal for a strong study is 80%.) Yes
  4.3. Were all enrolled subjects/patients (in the original sample) accounted for? Yes
  4.4. Were reasons for withdrawals similar across groups? Yes
  4.5. If diagnostic test, was decision to perform reference test not dependent on results of test under study? N/A
5. Was blinding used to prevent introduction of bias? Yes
  5.1. In intervention study, were subjects, clinicians/practitioners, and investigators blinded to treatment group, as appropriate? Yes
  5.2. Were data collectors blinded for outcomes assessment? (If outcome is measured using an objective test, such as a lab value, this criterion is assumed to be met.) Yes
  5.3. In cohort study or cross-sectional study, were measurements of outcomes and risk factors blinded? N/A
  5.4. In case control study, was case definition explicit and case ascertainment not influenced by exposure status? N/A
  5.5. In diagnostic study, were test results blinded to patient history and other test results? N/A
6. Were intervention/therapeutic regimens/exposure factor or procedure and any comparison(s) described in detail? Were interveningfactors described? Yes
  6.1. In RCT or other intervention trial, were protocols described for all regimens studied? Yes
  6.2. In observational study, were interventions, study settings, and clinicians/provider described? N/A
  6.3. Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect? Yes
  6.4. Was the amount of exposure and, if relevant, subject/patient compliance measured? Yes
  6.5. Were co-interventions (e.g., ancillary treatments, other therapies) described? N/A
  6.6. Were extra or unplanned treatments described? N/A
  6.7. Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups? N/A
  6.8. In diagnostic study, were details of test administration and replication sufficient? N/A
7. Were outcomes clearly defined and the measurements valid and reliable? Yes
  7.1. Were primary and secondary endpoints described and relevant to the question? Yes
  7.2. Were nutrition measures appropriate to question and outcomes of concern? Yes
  7.3. Was the period of follow-up long enough for important outcome(s) to occur? Yes
  7.4. Were the observations and measurements based on standard, valid, and reliable data collection instruments/tests/procedures? Yes
  7.5. Was the measurement of effect at an appropriate level of precision? Yes
  7.6. Were other factors accounted for (measured) that could affect outcomes? No
  7.7. Were the measurements conducted consistently across groups? Yes
8. Was the statistical analysis appropriate for the study design and type of outcome indicators? Yes
  8.1. Were statistical analyses adequately described and the results reported appropriately? Yes
  8.2. Were correct statistical tests used and assumptions of test not violated? Yes
  8.3. Were statistics reported with levels of significance and/or confidence intervals? Yes
  8.4. Was "intent to treat" analysis of outcomes done (and as appropriate, was there an analysis of outcomes for those maximally exposed or a dose-response analysis)? Yes
  8.5. Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)? Yes
  8.6. Was clinical significance as well as statistical significance reported? No
  8.7. If negative findings, was a power calculation reported to address type 2 error? N/A
9. Are conclusions supported by results with biases and limitations taken into consideration? Yes
  9.1. Is there a discussion of findings? Yes
  9.2. Are biases and study limitations identified and discussed? No
10. Is bias due to study's funding or sponsorship unlikely? N/A
  10.1. Were sources of funding and investigators' affiliations described? Yes
  10.2. Was the study free from apparent conflict of interest? Yes