Advanced Technology in Food Production

ATFP: Human Consumption of Plant Foods Produced Using Genetic Engineering (GE) Technologies (2015)


Morris J, Hawthorne KM, Hotze T, Abrams SA, Hirschi KD. Nutritional impact of elevated calcium transport activity in carrots. Proc Natl Acad Sci USA. 2008; 105 (5): 1,431-1,435.

PubMed ID: 18202180
Study Design:
Randomized Crossover Trial
A - Click here for explanation of classification scheme.
Quality Rating:
Neutral NEUTRAL: See Quality Criteria Checklist below.
Research Purpose:
To analyze the bioavailability of calcium in an engineered carrot that expresses increased levels of plant calcium.
Inclusion Criteria:
  • Healthy adults
  • Age 21 to 29.9 years.
Exclusion Criteria:
Not described.
Description of Study Protocol:


  • Public advertising
  • Word of mouth.


Randomized crossover trial.

Blinding Used

Implied with measurements.


  • Consumption of normal or sCAX1 carrots, modified to express increased levels of a plant calcium transporter (sCAX1); these plants contain two-fold-higher calcium content in the edible portions of the carrots
  • Calcium levels in plants were engineered through high-level expression of a deregulated Arabidopsis calcium transporter. An Arabidopsis vacuolar calcium antiporter, termed Cation exchanger 1 (CAX1), contains an N-terminal autoinhibitory domain. Expression of N-terminal truncations of CAX1 (sCAX1) in plants such as potatoes, tomatoes and carrots increases the calcium content in the edible portion of these foods. These sCAX1-expressing plants have heightened sequestration of calcium into the large central plant vacuoles.

Statistical Analysis

Statistical difference by ANOVA.

Data Collection Summary:

Timing of Measurements

  • Mice bones were analyzed at the end of feeding duration of six to seven weeks of age
  • Human urine samples were analyzed each day for a crossover study of two days.

Dependent Variables

  • 45Ca incorporation into mouse bone
  • 42Ca fractional absorption
  • Total calcium absorbed from 100g of carrots.

Independent Variables

sCAX1-expressing carrots or normal carrots.
Description of Actual Data Sample:
Initial N
  • Mice: N=120
  • Men: N=15
  • Women: N=15.
Characteristics of Human Subjects at Baseline

  Males Females
Age, years 24.2±1.6 25.7±2.8
Weight, kg 80.2±12.0 62.8±7.0
Height, cm 181.0±9.7 168.3±5.8
BMI, kg/m2 24.5±3.2 22.3±3.0
Ethnicity* 11W/1H/3A/0ME 11W/3H/0A/1ME
Calcium intake, mg per day 817±246 913±206

*W: White; H: Hispanic; A: Asian; ME: Multi-ethnic.


Houston, TX.

Summary of Results:

Key Findings

Mouse Feeding Study

  Control (1g) sCAX1-1 (0.5g) sCAX1-2 (0.5g)
45Ca incorporation (extrensic) 1.42±0.46%, 1.47±0.43% 1.66±0.55%, 1.7±0.52% 1.71±0.41%, 1.79±0.37%
45Ca incorporation (intrensic) 1.96±0.22%, 2.04±0.28% 1.95±0.32%, 2.03±0.33%  
 Human Feeding Study

  Control sCAX1 Statistical Significance
Calcium absorption efficiency 52.1±3.2% 42.6±2.8% P<0.001
Calcium absorption per 100g of carrot 26.50mg 15.34mg 41±2%

Other Findings

  • When people were fed sCAX1 and control carrots, total calcium absorption per 100g of carrots was 41±2% higher in sCAX1 carrots
  • Both the mice and human feeding studies demonstrate increased calcium absorption from sCAX1-expressing carrots compared with controls
  • Calcium incorporation by sCAX1 carrots was similar to the control carrots with 50% less carrots integrated into their feed.
Author Conclusion:
The findings establish unequivocally that modifying a single plant calcium transporter improves plant calcium absorption. These results demonstrate an alternative means of fortifying vegetables with bioavailable calcium.
Funding Source:
Government: US Department of Agriculture
University/Hospital: Baylor College, Texas A&M
Reviewer Comments:
  • Confounding factors such as supplement use, physical activity and baseline vitamin D levels of subjects were not taken into consideration
  • Small sample size of subjects; would benefit from a larger randomized control trial
  • Carrots were grown hydroponically, which is not standard practice for most carrots; variance of produce type and method of growth were not considered in impact on calcium absorption.
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? No
  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? No
  2.2. Were criteria applied equally to all study groups? Yes
  2.3. Were health, demographics, and other characteristics of subjects described? No
  2.4. Were the subjects/patients a representative sample of the relevant population? No
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.) N/A
  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? N/A
  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? ???
  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? No
  6.7. Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups? Yes
  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? Yes
  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)? N/A
  8.5. Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)? ???
  8.6. Was clinical significance as well as statistical significance reported? Yes
  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? Yes
10. Is bias due to study's funding or sponsorship unlikely? Yes
  10.1. Were sources of funding and investigators' affiliations described? Yes
  10.2. Was the study free from apparent conflict of interest? Yes