Advanced Technology in Food Production

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

Citation:

Nakamura R, Satoh R, Nakajima Y, Kawasaki N, Yamaguchi T, Sawada J, Nagoya H, Teshima R. Comparative study of GH-transgenic and non-transgenic amago salmon (Oncorhynchus masou ishikawae) allergenicity and proteomic analysis of amago salmon allergens. Regul Toxicol Pharmacol. 2009; 55 (3): 300-308.

PubMed ID: 19679156

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:

In this study, 1D-Western blotting with allergen-specific antibody was used to compare the expression of parvalbumin and fish type-1 collagen by non-GM and GH-transgenic amago salmons
Dual fluorescense-labeled 2D-Western blotting was used to accurately confirm the IgE-binding spots.

Inclusion Criteria:

GH-transgenic (GM, one-year) and non-transgenic amago salmons (non-GM, one- or two-year) were grown in the Fisheries Research agency of the National Research Institute of Aquaculture
Sera from 22 fish-allergic patients (eight from Japan and 14 from the United States) who were positive for fish-specific IgE, as well as sera from allergy-free persons used as a negative control
The study was approved by the Ethical Review Committee of the National Instute of Health Sciences.

Exclusion Criteria:

Not described.

Description of Study Protocol:

Recruitment: GH-transgenic (GM, one-year) and non-transgenic amago salmons (non-GM, one- or two-year) were grown in the Fisheries Research Agency of the National Research Institute of Aquaculture (Mie, Japan)
Design: Non-randomized crossover controlled trial
Blinding used: Implied with measurements
Intervention: The expression of parvalbumin and fish type-1 collagen by non-GM and GH-transgenic amago salmons were compared
Statistical analysis: Not described.

Data Collection Summary:

Timing of Measurements

All measurements made once.

Dependent Variables

1-D Western blot with patients' sera: The proteins in crude extracts of non-GM and GM-amago salmons were separated by SDS-PAGE on a 5% to 20% acrylamide gel and the proteins were transferred onto 0.22mcm nitrocellulose membranes
1-D Western blot with allergen-specific animal sera: The proteins in the crude extract from each of three bodies of non-GM (one-year and two-year) and GM amago (one-year) salmons were separated by SDS-Page on a 5% to 20% acrylamide gel and the proteins were transferred onto 0.22mcm nitrocellulose membranes
2D-PAGE and immunostaining with patients' sera or antiparvalbumin specific animal serum: Proteins were purified from a crude sample of amago salmon (10mcg) by using a 2D Clean-Up Kit, according to the manufacturer's instructions and the proteins were dissolved in 125mcL of Destreak Rehydration Solutions, supplemented with 0.1% IPG buffer
In-gel digestion: Proteins in crude extracts of amago salmon were separated by 2D-page and the protein spots corresponding to those that bound to patients IgE were cut from the gel. After destaining and drying them, the protein spots were incubated in reduction buffer.
Protein identification: The peptides were separated in an L-column Micro at a flow rate of 0.3mcL per minute in an HPLC system.

Independent Variables
Non-GM and GH-transgenic amago salmons.

Description of Actual Data Sample:

Initial N: 22 fish-allergic patients (eight from Japan and 14 from the United States) and three allergy-free controls
Attrition (final N): As above
Age: Not reported
Ethnicity: Not reported
Other relevant demographics: Not reported
Anthropometrics: Not reported
Location: Tokyo, Japan.

Summary of Results:

Key Findings

Western blotting with specific antibodies showed no increase in the content of the known allergens fish parvalbumin and fish-type I collagen in GM-amago salmon, in comparison with their content in non-GM amago salmon
The allergenome analysis of two fish-allergic patients allowed us to identify several IgE-binding proteins in amago salmon, including parvalbumin, triose-phosphate isomerase, fructose bisphosphate aldolase A and serum albumin, and there was no qualitative differences in these proteins between GM and non-GM amago salmons.

Author Conclusion:

The results of this study showed:

The major known fish allergens, parvalbumin and fish type-I collagen, were expressed by GM-amago salmon to a lesser degree or to the same degree as by non-GM-amago salmon
The serum IgE binding proteins parvalbumin (12kDA), triose-phosphate isomerase (26kDa), fructose bisphosphate aldolase (41kDA) and serum albumin (70kDa) were identified by proteomic analysis of amago salmon extracts
Proteomic analysis with patients' sera enabled the identification of overall variations in IgE-binding proteins, demonstrating the usefulness of proteomic analysis of samples containing unknown allergens
Analysis of sera from two fish-allergic patients showed that there were no qualitative differences in IgE-binding proteins between GM-amago salmon and non-GM-amago salmon.

Funding Source:

Government:

Grant from the Ministry of Health, Labour and Welfare

Reviewer Comments:

Only three control subjects
Fish-allergic patients were not well 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)	N/A
	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)	N/A

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?		???
	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?	No
	2.4.	Were the subjects/patients a representative sample of the relevant population?	???
3.	Were study groups comparable?		???
	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?	???
	3.3.	Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.)	???
	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?	N/A
	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?	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?	???
	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)?	N/A
	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?	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