Advanced Technology in Food Production

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

Citation:

Satoh R, Nakamura R, Komatsu A, Oshima M, Teshima R. Proteomic analysis of known and candidate rice allergens between non-transgenic and transgenic plants. Regul Toxicol Pharmacol. 2011; 59 (3): 437-444.

PubMed ID: 21300107

Study Design:

Non-Randomized Crossover Trial

Class:

C - Click here for explanation of classification scheme.

Quality Rating:

NEUTRAL: See Quality Criteria Checklist below.

Research Purpose:

The purpose of this study was to compare the qualitative and quantitative differences in known rice allergens between transgenic and non-transgenic rice, by using transgenic rice with high-level tryptophan (Trp) accumulation.

Inclusion Criteria:

Mature brown seeds of non-transgenic rice and OASA1D transgenic rice line HW5 harvested from an isolated field were used in the experiments
Sera from 16 Japanese allergy patients and 11 commercially available patients's sera or plasmas and control sera from three healthy volunteers were used in this study
Written informed consent was provided from all patients and volunteers.

Exclusion Criteria:

Not described.

Description of Study Protocol:

Recruitment

Obtainment of sera from 16 Japanese allergy patients and three healthy volunteers was unclear
11 patients' sera or plasmas were commercially available.

Design

Non-randomized crossover trial.

Blinding Used

Implied with measurements.

Intervention

Sera from allergic patients and healthy controls tested with proteins from non-transgenic and transgenic rice.

Statistical Analysis

Not described.

Data Collection Summary:

Timing of Measurements

Measurements made in all subjects.

Dependent Variables

Differences in rice allergens: The 19kDa, 52kDa and 63kDA rice globulin proteins that are candidate rice allergens were purified from the salt-soluble WT rice proteins. The concentrations of rabbit anti-rice globulin antibody-bound proteins were determined with a BCA protein assay reagent kit.
The salt-soluble proteins of WT rice and HW5 rice were separated by SDS-page on a 5% to 20% polyacrylamide gradient gel under reducing conditions and the separated proteins were transferred onto 0.22mcm nitrocellulose membranes
A 10mcg sample of rice protein was purified by using a 2D Cleanup Kit according to the manufacturer's protocol
For detection of 19kDa, 52kDa, and 63kDa rice globulin proteins, the protein-blotted membranes labeled with Cy5 mono-reactive dye and washed out the unbound Cy5 mono-reactive dye and washed out the unbound Cy5 with each antibody
A 200mcg sample of WT rice protein was separated by 2D page and stained with Pierce Stain for Mass Spectrometry. The silver-stained protein spots corresponding to the protein spots that bound to patients IgE were cut from the gel and in-gel digested for protein identification by MALDI-TOF-MS/MS.
Dot-blot immunoassays were performed by blotting four mcg of the anti-rabbit anti-rice globulin antibody-bound proteins onto nitrocellulose membranes. The bound IgE antibodies were detected by using Konica ImmunoStain HRP-1000, according to the manufacturer's protocol.

Independent Variables

Allergic patients versus healthy controls
Non-transgenic vs. transgenic rice.

Description of Actual Data Sample:

Initial N
Sera from 30 patients were used for this study:

Sera from 16 Japanese allergy patients (13 males, three females)
11 commercially available patients's sera or plasmas
Control sera from three healthy volunteers.

Attrition (Final N)
As above.

Age
Sera from 16 Japanese allergy patients ranged from ages one to 13 years.

Ethnicity
Assumed to be Japanese.

Other Relevant Demographics
Not described.

Anthropometrics
Not described.

Location
Japan.

Summary of Results:

Key Findings

1D immunoblot with antigen-specific-animal sera revealed no qualitative or quantitative differences in two known allergens, RAG2 and glyoxalase I, between non-transgenic and transgenic rice
Multiple spots containing known and novel IgE binding proteins were detected among the salt-soluble proteins of non-transgenic rice by 2D immunoblotting
Two globulin-like proteins a 52kDa protein and a 63kDa protein were identified as novel IgE-binding proteins that are candidates for rice allergens
These globulin-like proteins were homologous to Cupin superfamily allergens.

Author Conclusion:

In conclusion, the results from this study indicate that none of the known or novel endogenous IgE-binding proteins in this study appear to have been altered by genetic modification.

Funding Source:

Government:

Ministry of Health, Labour and Welfare of Japan

Not-for-profit

Food Safety Commission

Reviewer Comments:

Obtainment of some samples was unclear
Commercially available and control sera were not described
No statistical analysis was described.

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.)	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?	???
	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?	No
	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?	Yes
	6.6.	Were extra or unplanned treatments described?	Yes
	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?		No
	8.1.	Were statistical analyses adequately described and the results reported appropriately?	No
	8.2.	Were correct statistical tests used and assumptions of test not violated?	No
	8.3.	Were statistics reported with levels of significance and/or confidence intervals?	No
	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)?	No
	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?	No
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