Carrageenan and insulin resistance in humans: a randomised double-blind cross-over trial

Robert Wagner, Janine Buettner, Martin Heni, Louise Fritsche, Stephanie Kullmann, Moritz Wagmüller, Andreas Peter, Hubert Preissl, Jürgen Machann, Reiner Jumpertz von Schwartzenberg, Andreas L. Birkenfeld, Ulrich-Frank Pape, Gerrit van Hall, Peter Plomgaard, Hans-Ulrich Häring, Andreas Fritsche, Kelsey N. Thompson, Reinhild Klein, Norbert Stefan
BMC Medicine
BMC Medicine

Table of Contents

Overall Summary

Study Background and Main Findings

This study was a randomized, double-blind, placebo-controlled, cross-over trial investigating the effects of carrageenan on insulin resistance and related metabolic parameters in 20 young, non-obese men (mean age 27.4 ± 4.3 years, mean BMI 24.5 ± 2.5 kg/m2). Participants received either 250mg of carrageenan or a placebo twice daily for two weeks, with a washout period of 21-35 days between treatments. The primary outcomes were insulin sensitivity assessed by OGTT and hyperinsulinemic-euglycemic clamp. No significant differences were found between carrageenan and placebo for OGTT-based insulin sensitivity (p=0.52) or clamp-based insulin sensitivity (p=0.52). Secondary outcomes included hepatic insulin sensitivity, brain insulin sensitivity, hypothalamic inflammation, and hepatic triglyceride content, which also did not differ significantly between treatments. A significant interaction effect was found between BMI and treatment, with higher BMI associated with lower whole-body insulin sensitivity during the OGTT (p=0.04) and higher insulin resistance (HOMA2-IR; p=0.01) during carrageenan exposure. Carrageenan exposure significantly increased intestinal permeability, as measured by the lactulose-mannitol ratio (p=0.03) and plasma zonulin levels (placebo: 52.1 ng/l, carrageenan: 55.9 ng/l, p=0.05). In vitro assays demonstrated that carrageenan triggered the activation of CD19+ B cells and CD56+ NK cells and induced the production of pro-inflammatory cytokines (IL-6, IL-13, IL-17, TNF-beta, and GMCSF) in PBMCs.

Research Impact and Future Directions

This randomized, double-blind, placebo-controlled, cross-over trial in 20 young, non-obese men found that short-term carrageenan exposure (250mg twice daily for two weeks) did not significantly affect overall insulin sensitivity, measured by OGTT (p=0.52) and hyperinsulinemic-euglycemic clamp (p=0.52). However, a significant interaction between BMI and treatment was observed, with higher BMI associated with lower whole-body insulin sensitivity (OGTT, p=0.04), higher insulin resistance (HOMA2-IR, p=0.01), and a trend toward higher hypothalamic inflammation during carrageenan exposure. Carrageenan also significantly increased intestinal permeability, as measured by the lactulose-mannitol ratio (p=0.03) and plasma zonulin levels (p=0.05). These findings suggest a potential population impact where carrageenan may exacerbate metabolic dysfunction in individuals with higher BMI, potentially through mechanisms involving increased gut permeability and inflammation.

The study's findings suggest that implementation of dietary guidelines may need to consider the potential adverse effects of carrageenan, particularly in individuals with higher BMI. While the study did not directly assess dietary intake, the results suggest that reducing carrageenan consumption, especially in processed foods, could be a beneficial strategy for individuals at risk of metabolic disorders. This would require resources for public health education and potentially food labeling regulations.

There are uncertainties regarding the long-term effects of carrageenan exposure and the specific thresholds of BMI at which the adverse effects become clinically significant. The study's findings suggest a potential risk of increased insulin resistance and inflammation in individuals with higher BMI, but the benefits of avoiding carrageenan need to be weighed against the potential costs and challenges of altering dietary habits. Subgroup considerations are crucial, as the study suggests that the effects of carrageenan may be more pronounced in individuals with pre-existing metabolic vulnerability.

Key knowledge gaps remain regarding the long-term effects of carrageenan consumption, the specific mechanisms underlying the BMI-dependent effects, and the role of the gut microbiome. The study's limitations, including the small sample size, the recruitment of only young, healthy males, and the short exposure period, may underestimate the true effect size of carrageenan in a broader population. Future research should focus on larger, more diverse populations, longer exposure periods, and a more detailed investigation of the gut microbiome's role. Clinically, the findings suggest a need for caution regarding carrageenan intake, particularly in individuals with higher BMI, and highlight the importance of further research to inform dietary recommendations.

Critical Analysis and Recommendations

Clear Research Question (written-content)
The abstract clearly states the research question, focusing on carrageenan's impact on insulin resistance and related metabolic parameters in humans; This is a well-defined and clinically relevant research question, addressing a gap in knowledge regarding the effects of a common food additive; This clarity allows for focused investigation and interpretation of results; The clear research question facilitates the translation of findings into potential public health recommendations.
Section: Abstract
Concise Summary of Study Design (written-content)
The abstract concisely summarizes the study design: randomized, double-blind, placebo-controlled, cross-over trial with 20 male participants (age 27.4 ± 4.3 years, BMI 24.5 ± 2.5 kg/m2), and intervention (250mg carrageenan or placebo twice daily for two weeks); This design is robust for investigating intervention effects, minimizing bias and controlling for individual variability; The concise summary allows readers to quickly grasp the study's methodology; This design enhances the internal validity of the study and the reliability of the findings.
Section: Abstract
Presentation of Main Findings (written-content)
The abstract presents the main findings: no significant overall difference in insulin sensitivity, but a significant interaction between BMI and treatment (OGTT-based insulin sensitivity index: p=0.04, fasting insulin resistance: p=0.01, hepatic insulin sensitivity index: p=0.04); This highlights the key result that carrageenan's effects may depend on BMI; Presenting both the overall null effect and the interaction is crucial for a balanced interpretation; This finding suggests a need for personalized dietary recommendations based on BMI.
Section: Abstract
Clarify Direction of BMI Interaction (written-content)
The abstract should specify the direction of the BMI interaction, stating that higher BMI was associated with lower insulin sensitivity during carrageenan exposure; This lack of directionality limits the immediate interpretability of the key finding; Clarifying the direction is crucial for understanding the clinical implications; Adding this detail would significantly enhance the abstract's informativeness.
Section: Abstract
Clear Public Health Context (written-content)
The background section clearly introduces the public health context, highlighting the global rise in type 2 diabetes and its connection to the 'Western-style' diet, characterized by ultra-processed foods; This establishes the relevance and importance of the research question; Connecting the research to a major public health issue strengthens the study's rationale; This context helps to justify the need for investigating specific dietary factors like carrageenan.
Section: Background
Effective Introduction of Carrageenan (written-content)
The section effectively introduces carrageenan, describing its widespread use, chemical structure, and the distinction between high molecular-weight carrageenan and poligeenans; This provides necessary background information for understanding the study's focus; Defining carrageenan and its different forms is crucial for interpreting the results; This information helps readers to understand the potential mechanisms of action.
Section: Background
Quantify Typical Carrageenan Intake (written-content)
The section should provide a range or estimate of typical daily carrageenan intake in Western diets; The lack of this information makes it difficult to assess the relevance of the study's intervention dose; Quantifying typical intake is crucial for contextualizing the study's findings; Adding this detail would improve the background section's completeness.
Section: Background
Appropriate Study Design (written-content)
The study employed a placebo-controlled, randomized, double-blind, cross-over design; This design is a rigorous approach, minimizing bias and controlling for inter-individual variability; The design enhances the internal validity of the study; This rigorous design strengthens the confidence in the study's findings.
Section: Methods
Rigorous Insulin Sensitivity Measurement (written-content)
The study used both OGTT and hyperinsulinemic-euglycemic clamp to assess insulin sensitivity, the latter being the gold standard; This rigorous measurement approach adds to the study's methodological strength; Using the gold standard enhances the validity and reliability of the insulin sensitivity measurements; This strengthens the confidence in the study's findings regarding insulin sensitivity.
Section: Methods
Specify Carrageenan Type (written-content)
The Methods section should specify the type of carrageenan used (kappa, iota, or lambda); Different types have different properties and potentially different biological effects; This detail is crucial for interpreting the results and for replication; Specifying the type would enhance the study's transparency and reproducibility.
Section: Methods
Assess and Report Intervention Compliance (written-content)
The Methods section should state whether and how compliance with the intervention was assessed; Knowing whether participants took the capsules as prescribed is crucial for interpreting the results; Assessing compliance addresses a potential source of bias; Reporting compliance would strengthen the study's methodological rigor.
Section: Methods
Clear Presentation of Primary Outcome (written-content)
The Results section clearly presents the main finding: overall insulin sensitivity did not differ significantly between treatments (OGTT: p=0.52, n=20 pairs; clamp: p=0.52, n=19 pairs); This direct presentation of the primary outcome is crucial for a Results section; Providing the p-values and sample sizes allows for assessment of statistical significance; This clear presentation facilitates understanding of the main result.
Section: Results
Highlighting Increased Intestinal Permeability (written-content)
Carrageenan exposure significantly increased intestinal permeability, as evidenced by an elevated lactulose-mannitol ratio (p=0.03, n=19) and higher plasma zonulin levels (55.9 vs 52.1 ng/l, p=0.05, n=20); This finding suggests that carrageenan may disrupt gut barrier function; Providing the p-values and sample sizes allows for assessment of statistical significance; This finding is important for understanding the potential mechanisms of carrageenan's effects.
Section: Results
Reporting of BMI x Treatment Interactions (written-content)
Interaction analyses showed lower whole-body insulin sensitivity (OGTT, p=0.04) and higher insulin resistance (HOMA2-IR, p=0.01) for carrageenan with higher BMI; This suggests that the effects of carrageenan on insulin sensitivity may be dependent on BMI; Providing the p-values allows for assessment of statistical significance; This is a key finding that modifies the interpretation of the primary outcome.
Section: Results
Report Actual Values for Outcomes (written-content)
The Results section must provide the actual values (means and standard deviations/errors) for the primary and secondary outcomes, not just p-values; Without these values, the magnitude and direction of any effect (or lack thereof) are unknown; This is essential for assessing clinical relevance and for comparing results to other studies; Including these values is crucial for the clarity and interpretability of the Results section.
Section: Results
Report Carry-Over Effect Test Results (written-content)
The Results section must report the results of the statistical tests for carry-over effects for each outcome; This is crucial for assessing the validity of the cross-over design; Without this information, the reliability of the findings is questionable; Reporting these results is necessary for the completeness and accuracy of the Results section.
Section: Results
Effective Summary of Main Findings (written-content)
The Discussion effectively summarizes the main findings, reiterating the lack of overall effect on insulin sensitivity and the significant interaction with BMI; This provides a concise overview of the study's key results; Summarizing the main findings is essential for reinforcing the study's conclusions; This helps readers to understand the overall implications of the study.
Section: Discussion
Contextualization within Existing Literature (written-content)
The section appropriately places the findings in the context of existing literature, citing relevant studies on carrageenan's effects in animal models and humans; This helps to connect the study's findings to the broader body of knowledge; Contextualizing the findings is important for assessing their novelty and significance; This strengthens the study's contribution to the field.
Section: Discussion
Reconcile Overall Null Effect with BMI Interaction (written-content)
The Discussion should more explicitly discuss the discrepancy between the lack of overall effect on insulin sensitivity and the significant interaction with BMI; These two findings are presented, but not fully reconciled; A more thorough discussion of why carrageenan might affect insulin sensitivity only in individuals with higher BMI is needed; This would provide a more nuanced and insightful interpretation.
Section: Discussion
Address Clinical Significance of Increased Permeability (written-content)
The Discussion should more directly address the clinical significance of the observed increase in intestinal permeability; While the section describes the mechanisms, it doesn't fully explain the practical implications of the magnitude of increase observed; Explaining the clinical relevance is crucial for translating the findings into potential health recommendations; Adding this discussion would strengthen the study's impact.
Section: Discussion

Section Analysis

Abstract

Key Aspects

Strengths

Suggestions for Improvement

Non-Text Elements

Table S1 Characteristics of the study participants at randomization
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Table S1 Characteristics of the study participants at randomization
First Reference in Text
No explicit numbered reference found
Description
  • Overview of Table S1 and its components: Table S1 presents the baseline characteristics of the study participants at randomization. Randomization is the process of assigning participants to different treatment groups by chance, with the goal of creating groups that are similar in terms of their characteristics at the start of the study. This table shows how similar the two treatment groups (carrageenan-first and placebo-first) were at baseline. The table includes the following characteristics: age, height, weight, BMI (Body Mass Index), waist circumference, hip circumference, waist-to-hip ratio, and body surface area. For each characteristic, the table presents the mean (average) and standard deviation for each treatment group, as well as a p-value. The p-value is a measure of statistical significance, which indicates the likelihood that the observed difference between the two groups is due to chance.
Scientific Validity
  • Importance of baseline characteristics and interpretation of p-values: The presentation of baseline characteristics is essential for assessing the validity of a clinical trial. This table allows the reader to evaluate whether the randomization process was successful in creating comparable treatment groups. The inclusion of p-values is appropriate for assessing whether there are any statistically significant differences between the groups at baseline. However, it's important to note that statistical significance does not necessarily imply clinical significance. Even if there are no statistically significant differences between the groups at baseline, it's still possible that there are clinically meaningful differences that could affect the results of the study.
  • Comprehensiveness of baseline characteristics: The table includes a comprehensive set of baseline characteristics that are relevant to the study outcomes. However, it would be helpful to include additional characteristics that could potentially influence the results, such as dietary habits, physical activity levels, and medical history. Including information about the racial and ethnic composition of the sample would further enhance the table.
Communication
  • Clarity and organization of the table: The table is well-organized and easy to read. The use of clear column headings and consistent formatting makes it easy to compare the characteristics of the two treatment groups. The inclusion of p-values allows the reader to quickly assess whether there are any statistically significant differences between the groups at baseline.

Background

Key Aspects

Strengths

Suggestions for Improvement

Methods

Key Aspects

Strengths

Suggestions for Improvement

Non-Text Elements

Figure S1 Participant flow in the study
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Figure S1 Participant flow in the study
First Reference in Text
The flow of the study is shown in Fig. S1.
Description
  • Overview of the participant flow diagram: Figure S1 is a participant flow diagram, also known as a CONSORT diagram, which visually summarizes the progress of participants through a clinical trial. It starts with the number of individuals assessed for eligibility (n=28). Eligibility refers to the criteria that individuals must meet to be included in the study. It then shows the number of individuals who were excluded and the reasons for their exclusion (n=4). The reasons for exclusion include not meeting the inclusion criteria (n=1), refusing to participate (n=2), and other reasons (n=1). Next, it shows the number of individuals who were randomized (n=24). Randomization is the process of assigning participants to different treatment groups by chance. In this case, participants were randomized to either the carrageenan-first group or the placebo-first group. The diagram then shows the number of individuals in each treatment group (n=12 in each group). It also shows the number of individuals who were lost to follow-up and the reasons for their dropout. For the carrageenan-first group, the reasons for dropout were personal reasons (n=1) and appointment conflicts (n=1). For the placebo-first group, the reasons for dropout were protocol violation (n=1) and consent withdrawn (n=1). Finally, it shows the number of individuals who were analyzed in each treatment group (n=10 in each group).
Scientific Validity
  • Adherence to CONSORT guidelines and completeness of information: The participant flow diagram adheres to CONSORT guidelines, which are a set of recommendations for reporting clinical trials. This ensures that the diagram provides a complete and transparent overview of the study's participant flow. The diagram includes all key stages of the trial, including assessment for eligibility, randomization, treatment allocation, follow-up, and analysis. The reasons for exclusion and dropout are clearly stated, which is important for assessing the potential for bias.
  • Importance of the diagram for assessing bias and generalizability: The participant flow diagram provides essential information for assessing the validity and generalizability of the study's findings. By showing the number of participants excluded and lost to follow-up, the diagram helps to assess the potential for selection bias and attrition bias. The reasons for exclusion and dropout can also provide insights into the characteristics of the study population and the potential limitations of the findings.
Communication
  • Clarity and organization of the flow diagram: The flow diagram is clearly laid out and easy to follow. The use of boxes and arrows effectively illustrates the progression of participants through the different stages of the study. The reasons for exclusion and dropout are clearly stated, which is important for transparency.
Figure S2 Scheme of the randomized controlled crossover design.
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Figure S2 Scheme of the randomized controlled crossover design.
First Reference in Text
The participants were randomised to the treatments (Fig. S2) using a computerised block-randomisation with a block-size of 10.
Description
  • Overview of the crossover design: Figure S2 shows the design of the randomized controlled crossover trial. A crossover trial is a type of clinical trial where participants receive a sequence of different treatments (or exposures). This figure shows how the participants are exposed to the two treatments (placebo or carrageenan). The figure shows the timeline of the study with the following visits. V0 is the screening visit to determine if the participant is eligible. V1 is the start of the study medication. Visits V2, V3 and V4 are the assessment visits (Block A) after the first treatment phase. V5 is the start of the study medication in the second phase. Visits V6, V7 and V8 are the assessment visits (Block B) after the second treatment phase. The figure indicates that there is a 14-day exposition phase, followed by a 21-35 day washout period, followed by another 14-day exposition phase. The order of treatments is randomized.
  • Key information about randomization from the reference text: The reference text states that participants were randomized to the treatments using a computerized block-randomization with a block size of 10. Block randomization is a method used to ensure that the number of participants in each treatment group is balanced over time. A 'block' is a predetermined number of participants (in this case, 10) who will be randomized together. This ensures that after every 10 participants, there will be an equal number of participants in each treatment group. The block size helps to maintain balance even if there are unforeseen events that cause participants to drop out of the study.
Scientific Validity
  • Appropriateness of the study design and randomization method: The use of a randomized controlled crossover design is a strength of the study, as it allows for the assessment of within-subject effects and reduces the potential for confounding. The description of the block randomization method is appropriate. Block randomization helps to ensure balance in treatment assignments, particularly in smaller trials. The choice of a block size of 10 is reasonable.
  • Importance of the washout period and justification for its duration: The figure clearly shows the sequence of treatments and the washout period. The washout period is important to allow the effects of the first treatment to dissipate before the second treatment is administered. The duration of the washout period (21-35 days) appears reasonable, but it would be helpful to provide justification for this choice in the methods section. The study should confirm the half-life of carrageenan is substantially less than the washout period, and that there is no plausible mechanism for carryover effects after this interval.
Communication
  • Clarity and completeness of the figure: The figure clearly illustrates the sequence of events in the crossover design, including the exposition phases, washout period, and assessment visits. The labeling of the visits (V0, V1, V2, etc.) is helpful for understanding the timeline. However, the figure could benefit from explicitly stating the duration of each assessment visit (V2,3,4 and V6,7,8) to provide a more complete picture of the study design.

Results

Key Aspects

Strengths

Suggestions for Improvement

Non-Text Elements

Fig. 1 A-C Differences in whole-body insulin sensitivity (A) and organ-related...
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Fig. 1 A-C Differences in whole-body insulin sensitivity (A) and organ-related insulin sensitivity (predominantly skeletal muscle (B), liver (C)) after placebo (PCB) and carrageenan (CGN) administration in the study.

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Fig. 1 A-C Differences in whole-body insulin sensitivity (A) and organ-related insulin sensitivity (predominantly skeletal muscle (B), liver (C)) after placebo (PCB) and carrageenan (CGN) administration in the study.
First Reference in Text
None of these variables showed differences between treatments (n=20 pairs for the OGTT and n=19 pairs for clamp, p=0.52 for both, Fig. 1A, B).
Description
  • Overview of Figure 1 and its components: Figure 1 presents the effects of placebo versus carrageenan on insulin sensitivity. Insulin sensitivity refers to how responsive your body is to insulin. Insulin is a hormone that helps glucose (sugar) from the food you eat enter your cells for energy. If you are insulin sensitive, it means your body needs less insulin to lower blood glucose levels. The figure is split into three parts (A, B, and C), each showing different measures of insulin sensitivity. Part A shows whole-body insulin sensitivity as measured by OGTT, or oral glucose tolerance test. An OGTT involves drinking a sugary drink and then measuring blood glucose levels over time. Part B shows whole-body insulin sensitivity as measured by a clamp, which is a more direct measure that involves infusing insulin and glucose to maintain a constant blood glucose level. Part C shows hepatic insulin sensitivity, which is how sensitive the liver is to insulin. The data is presented as box plots, which show the median (the middle value), the interquartile range (the range of the middle 50% of the data), and the range of the data. Dashed lines connect individual data points to show changes within each participant after placebo versus carrageenan. The p-values, which are a measure of statistical significance, are shown for each graph.
  • Key statistical results from the reference text: The reference text indicates that there were no significant differences observed between the placebo and carrageenan treatments for whole-body insulin sensitivity (OGTT: p=0.52, n=20 pairs; clamp: p=0.52, n=19 pairs). The number of pairs (n) indicates the number of participants for whom data was available for both the placebo and carrageenan conditions. The p-value of 0.52 suggests that the observed differences between the two treatments are likely due to random chance rather than a real effect of carrageenan.
Scientific Validity
  • Appropriateness of statistical analysis and interpretation: The statistical analysis appears to be appropriate for the study design, using paired t-tests to compare within-subject differences. The reporting of p-values and sample sizes is consistent with standard practice. However, the relatively high p-values (0.52) suggest a lack of statistical power to detect potential differences, which should be acknowledged. It would be beneficial to include effect sizes and confidence intervals to provide a more complete picture of the results.
  • Appropriateness of data visualization: The figure displays paired data, which is appropriate for a crossover study design. This allows for the assessment of within-subject changes. The use of box plots is a reasonable choice for visualizing the distribution of the data. However, the lack of statistically significant differences should be interpreted cautiously, as it does not necessarily indicate the absence of a real effect.
Communication
  • Clarity and completeness of the caption: The figure caption clearly identifies the figure's content, specifying the variables examined (insulin sensitivity) and the experimental conditions (placebo vs. carrageenan). The use of abbreviations (PCB and CGN) is helpful for brevity, especially since they are defined in the caption itself. However, it might be useful to explicitly state in the caption that the graphs display paired data to further clarify that each subject received both treatments.
Fig. 2 A, B Intestinal permeability expressed by lactulose-mannitol ratio (A)...
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Fig. 2 A, B Intestinal permeability expressed by lactulose-mannitol ratio (A) and plasma zonulin levels (B) after treatments with placebo (PCB) and carrageenan (CGN).

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Fig. 2 A, B Intestinal permeability expressed by lactulose-mannitol ratio (A) and plasma zonulin levels (B) after treatments with placebo (PCB) and carrageenan (CGN).
First Reference in Text
There was no difference in C-reactive protein (CRP) and interleukin-6 (IL-6) levels between the treatments (both p>0.5). No carry-over effects were detected (p>0.05).
Description
  • Overview of Figure 2 and its components: Figure 2 illustrates the effects of placebo versus carrageenan on intestinal permeability. Intestinal permeability, sometimes called "leaky gut," refers to how easily substances can pass through the lining of the small intestine into the bloodstream. A healthy intestine has tight junctions that control what gets in and out. When these junctions become loose, larger molecules, bacteria, and toxins can leak through, potentially triggering inflammation and other health problems. Part A shows intestinal permeability as measured by the lactulose-mannitol ratio. This test involves drinking a solution containing lactulose and mannitol, two sugars with different molecular sizes. The amount of each sugar that is recovered in the urine is measured. Since lactulose is larger, it normally doesn't pass through the intestinal lining as easily as mannitol. A higher lactulose-mannitol ratio indicates increased intestinal permeability because more of the larger lactulose molecule is passing through. Part B shows intestinal permeability as measured by plasma zonulin levels. Zonulin is a protein that regulates the tight junctions in the intestinal lining. When zonulin levels are elevated, it can lead to increased intestinal permeability. The data is presented as box plots, which show the median, interquartile range, and range of the data. Dashed lines connect individual data points to show changes within each participant after placebo versus carrageenan. The p-values are shown for each graph.
  • Key statistical results from Figure 2: Figure 2A shows that the lactulose-mannitol ratio is significantly higher after carrageenan treatment compared to placebo (p=0.03, n=19 pairs). This indicates that carrageenan increases intestinal permeability. Figure 2B shows that plasma zonulin levels are also significantly higher after carrageenan treatment compared to placebo (p=0.05, n=20 pairs), further supporting the conclusion that carrageenan increases intestinal permeability. The number of pairs (n) indicates the number of participants for whom data was available for both conditions. The p-values indicate the level of statistical significance.
  • Key statistical results from the reference text: The reference text states that there was no difference in C-reactive protein (CRP) and interleukin-6 (IL-6) levels between the treatments (both p>0.5). These are markers of systemic inflammation. The lack of significant difference suggests that carrageenan does not cause a detectable increase in systemic inflammation, at least as measured by these two markers. The reference text also indicates that no carry-over effects were detected (p>0.05), which is important for the validity of the crossover study design.
Scientific Validity
  • Appropriateness of methodology and statistical analysis: The use of the lactulose-mannitol test is a well-established method for assessing intestinal permeability. The measurement of plasma zonulin levels provides additional support for the findings. The statistical analysis appears appropriate, and the reporting of p-values and sample sizes is consistent with standard practice. The significant p-values (0.03 and 0.05) suggest that carrageenan has a statistically significant effect on intestinal permeability and zonulin levels. The lack of carryover effects strengthens the validity of the results.
  • Interpretation of results in the context of other findings: The lack of significant differences in CRP and IL-6 levels, as noted in the reference text, does not necessarily contradict the findings of increased intestinal permeability. Intestinal permeability can lead to localized inflammation in the gut without necessarily causing a detectable increase in systemic inflammation. It would be helpful to discuss the potential mechanisms by which carrageenan might increase intestinal permeability and whether the observed changes in zonulin levels are consistent with these mechanisms.
Communication
  • Clarity and completeness of the caption: The caption clearly identifies the figure's content, specifying the measures of intestinal permeability (lactulose-mannitol ratio and plasma zonulin levels) and the experimental conditions (placebo vs. carrageenan). The use of abbreviations (PCB and CGN) is helpful, especially since they are defined in previous captions. It might be helpful to briefly mention in the caption that an increased lactulose-mannitol ratio and increased zonulin levels indicate higher intestinal permeability for readers unfamiliar with these measures.
Fig. 3 A-F Interaction between BMI and treatment (placebo-PCB, carrageenan-CGN)...
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Fig. 3 A-F Interaction between BMI and treatment (placebo-PCB, carrageenan-CGN) on predefined study endpoints.

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Fig. 3 A-F Interaction between BMI and treatment (placebo-PCB, carrageenan-CGN) on predefined study endpoints.
First Reference in Text
These interaction analyses showed lower whole-body insulin sensitivity during the OGTT (p=0.04, Fig. 3A) and higher insulin resistance estimated from fasting glucose and insulin levels (HOMA2-IR; p=0.01, Fig. 3B) for carrageenan with higher BMI.
Description
  • Overview of Figure 3 and its components: Figure 3 explores how the effect of carrageenan might differ depending on a person's Body Mass Index (BMI). BMI is a measure of body fat based on height and weight, used to classify individuals as underweight, normal weight, overweight, or obese. An 'interaction' in this context means that the effect of carrageenan on a particular outcome (like insulin sensitivity) is not the same for everyone, but changes depending on their BMI. The figure presents six different graphs (A-F), each showing the relationship between BMI and a different study endpoint, separately for the placebo and carrageenan treatments. These endpoints include: A) whole-body insulin sensitivity as measured by OGTT, B) insulin resistance as measured by HOMA2-IR, C) insulin sensitivity of glucose disposal (clamp), D) hepatic insulin sensitivity, E) C-reactive protein (a marker of inflammation), and F) hypothalamic inflammation. Each graph shows data points for individual participants, as well as regression lines that represent the average relationship between BMI and the endpoint for each treatment group. These lines allow one to visualize how the effect of carrageenan varies across different BMI levels.
  • Key statistical results from the reference text: The reference text highlights two key findings from the interaction analyses. First, it states that there was lower whole-body insulin sensitivity during the OGTT (p=0.04) for carrageenan treatment in individuals with higher BMI. This means that, among people with higher BMI, carrageenan appeared to worsen their body's response to insulin. Second, it states that there was higher insulin resistance estimated from fasting glucose and insulin levels (HOMA2-IR; p=0.01) for carrageenan with higher BMI. HOMA2-IR is a calculation that estimates how resistant the body is to the effects of insulin, with higher numbers indicating greater resistance. These results suggest that the negative effects of carrageenan on insulin sensitivity are more pronounced in people with higher BMI.
Scientific Validity
  • Appropriateness of statistical analysis and interpretation: The use of interaction analyses is appropriate for investigating whether the effect of carrageenan varies depending on BMI. The statistical method used (mixed linear regression models) is suitable for analyzing this type of data. The reporting of p-values is consistent with standard practice. However, it's important to note that correlation does not equal causation. The observed interactions suggest an association between BMI, carrageenan treatment, and insulin sensitivity, but further research is needed to establish a causal relationship. The relatively small p-values (0.04 and 0.01) suggest statistical significance, but the clinical significance should also be considered.
  • Limitations of interpretation and generalizability: The interpretation of the interaction effects should be cautious. While the interaction plots provide a visual representation of the relationship between BMI, treatment, and endpoints, it's important to consider the limitations of regression models. Extrapolation beyond the observed range of BMI values should be avoided. Furthermore, the study population consists of young, non-obese males, which limits the generalizability of the findings to other populations.
Communication
  • Clarity and completeness of the caption: The caption clearly indicates that the figure explores the interaction between Body Mass Index (BMI) and the treatment (placebo or carrageenan) on various study endpoints. The use of abbreviations is consistent with previous figures. However, the caption could benefit from briefly listing the specific endpoints visualized in panels A-F to give the reader a clearer idea of the figure's content at a glance.
  • Visual clarity of interaction plots: The interaction plots are generally well-labeled, but the visual representation of the interaction could be improved. Consider using different colors or line styles to better distinguish between the placebo and carrageenan groups. Adding confidence intervals around the regression lines would also provide a better sense of the uncertainty in the estimates.
Fig. 4 A, B In vitro analysis of peripheral blood mononuclear cells (PBMCs)...
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Fig. 4 A, B In vitro analysis of peripheral blood mononuclear cells (PBMCs) from 15 healthy individuals before carrageenan exposure.

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Fig. 4 A, B In vitro analysis of peripheral blood mononuclear cells (PBMCs) from 15 healthy individuals before carrageenan exposure.
First Reference in Text
Furthermore, induction of the cytokines IL-6, IL-13, IL-17, TNF-beta, and GMCSF was observed (Fig. 4).
Description
  • Overview of Figure 4 and its components: Figure 4 shows the results of experiments where peripheral blood mononuclear cells (PBMCs) were taken from 15 healthy people, then exposed to carrageenan in a petri dish. PBMCs are a mix of different types of immune cells, including T cells, B cells, and natural killer (NK) cells. Instead of giving the carrageenan to the people and then taking out their blood, this experiment added the carrageenan directly to the cells in a dish. Part A of the figure shows the effect of carrageenan on the proliferation (growth) of PBMCs, as well as the activation of T cells, B cells, and NK cells. Activation means that these cells are turned 'on' and are starting to do their job in the immune system. Activation is measured by looking for a marker called CD69 on the surface of the cells; cells with CD69 are considered activated. Part B of the figure shows the effect of carrageenan on the production of cytokines. Cytokines are signaling molecules that immune cells use to communicate with each other. They can be pro-inflammatory (promoting inflammation) or anti-inflammatory (reducing inflammation). The figure shows the levels of various cytokines produced by the PBMCs after exposure to carrageenan.
  • Key statistical results from the reference text: The reference text indicates that the researchers observed the induction (increase) of the cytokines IL-6, IL-13, IL-17, TNF-beta, and GMCSF. These are all pro-inflammatory cytokines, meaning they promote inflammation. This suggests that carrageenan can directly stimulate immune cells to release inflammatory signals.
Scientific Validity
  • Appropriateness of methodology and interpretation: The use of in vitro experiments is a valid approach for investigating the direct effects of carrageenan on immune cells. The use of PBMCs is relevant, as these cells play a key role in the immune response. However, it's important to note that in vitro experiments do not fully replicate the complex environment of the human body. Therefore, the results should be interpreted cautiously and in the context of the in vivo findings.
  • Missing information about experimental conditions and statistical analysis: It is not clear from the figure caption or text what concentrations of carrageenan were used in the in vitro experiments. This information is essential for interpreting the results and comparing them to other studies. It is also unclear what statistical methods were used to analyze the data. This information should be provided in the figure caption or methods section.
  • Consistency with previous research and limitations of the study population: The finding that carrageenan induces the production of pro-inflammatory cytokines is consistent with previous research. However, it's important to consider the potential limitations of using PBMCs from healthy individuals. The response of PBMCs from individuals with underlying inflammation or other health conditions might be different.
Communication
  • Clarity and completeness of the caption: The caption clearly states that the figure presents an in vitro analysis of PBMCs from healthy individuals before they were exposed to carrageenan in vivo. This clarifies that the PBMCs were exposed to carrageenan directly in the lab, not taken from participants after they consumed carrageenan. However, the caption could be more specific about what aspects of PBMCs are being analyzed (e.g., activation and cytokine production).
  • Visual clarity of bar graphs: The figure uses bar graphs to present the data. The graphs are generally well-labeled, but it would be helpful to include error bars (e.g., standard error of the mean) to provide a sense of the variability in the data. It is also not clear whether the data is presented as absolute numbers or fold change.
Figure S3 Interindividual diversity in stool microbiome samples obscures any...
Full Caption

Figure S3 Interindividual diversity in stool microbiome samples obscures any potential impact of carrageen use on the composition of the gut microbiome.

Figure/Table Image (Page 15)
Figure S3 Interindividual diversity in stool microbiome samples obscures any potential impact of carrageen use on the composition of the gut microbiome.
First Reference in Text
For each individual, their top representative taxa in both exposition phases (blocks A and B) of the study are visualised (Fig. S3).
Description
  • Overview of Figure S3 and its components: Figure S3 presents data on the gut microbiome composition of the study participants. The gut microbiome is the collection of all microorganisms (bacteria, fungi, viruses, etc.) that live in the digestive tract. These microorganisms play an important role in human health. The figure is designed to investigate whether carrageenan use has an impact on the composition of the gut microbiome. Panel A presents bar plots showing the relative abundance of the top representative taxa (groups of organisms) for each individual in both exposition phases (Block A and Block B). Relative abundance refers to the proportion of each taxon within the total microbial community. Each bar represents an individual, and the different colors within each bar represent the different taxa. Panel B presents a Principal Coordinates Analysis (PCoA) plot. PCoA is a technique used to visualize the similarity between different samples based on their microbiome composition. Each point on the plot represents a sample, and samples that are closer together are more similar in terms of their microbiome composition. The plot shows samples from both treatment groups (placebo and carrageenan) and both time points (Block A and Block B). Panel C presents univariate R-squared values for various clinical covariates.
  • Key information from the reference text: The reference text indicates that the figure visualizes the top representative taxa for each individual in both exposition phases (blocks A and B) of the study. It is meant to show the diversity in microbiome composition between individuals.
Scientific Validity
  • Appropriateness of methodology and statistical analysis: The use of 16S rRNA gene sequencing and bioinformatic analysis is a standard approach for characterizing the gut microbiome composition. The use of PCoA is also appropriate for visualizing the similarity between samples. However, it's important to note that 16S rRNA gene sequencing only provides information about the bacterial composition of the microbiome and does not capture the diversity of other microorganisms, such as fungi and viruses. The figure does not present any statistical analyses to support the conclusion that carrageenan has no impact on the gut microbiome. While the figure suggests that interindividual diversity obscures any potential effects, statistical tests should be performed to confirm this.
  • Limitations of snapshot analysis and lack of information about microbiome stability: The figure provides a snapshot of the gut microbiome composition at two time points. However, the gut microbiome is a dynamic ecosystem that can change rapidly in response to various factors, such as diet and lifestyle. Therefore, it's possible that carrageenan has transient effects on the gut microbiome that are not captured by this analysis. The figure does not provide information about the stability of the gut microbiome composition over time. Assessing the stability of the microbiome would strengthen the analysis.
Communication
  • Clarity and informativeness of the caption: The caption accurately reflects the overall finding that interindividual variation in the gut microbiome makes it difficult to detect any specific effects of carrageenan. However, the caption could be more informative by briefly mentioning the types of analyses presented in the figure (e.g., bar plots of taxa abundance, PCoA plot).
  • Visual clarity and labeling of the figure panels: The figure consists of multiple panels (A, B, and C). It may not be immediately clear to the reader what each panel represents. Clearer titles for each panel would improve the understanding of the figure. The color scheme in the bar plots (Panel A) is difficult to discern, making it hard to compare the relative abundance of different taxa. A more distinct color palette would be beneficial.
Table S2 Study endpoints after placebo and carrageenan expositions
Figure/Table Image (Page 17)
Table S2 Study endpoints after placebo and carrageenan expositions
First Reference in Text
Results for all pre-specified study endpoints are shown in Table S2.
Description
  • Overview of Table S2 and its components: Table S2 presents the results for all pre-specified study endpoints after the placebo and carrageenan expositions. An 'endpoint' in a clinical trial is a specific outcome that is measured to see if the treatment had an effect. The table compares the values of these endpoints after the participants received either the placebo or carrageenan. The table includes the following information for each endpoint: the number of participants in each group (n1 and n2), the mean (average) value for each group, the standard error of the mean, and a p-value. The p-value indicates whether the difference between the two groups is statistically significant. The table includes a wide range of endpoints related to insulin sensitivity, glucose metabolism, inflammation, and gut permeability.
  • Key statistical results from Table S2: The results presented are mean ± standard errors for each group. The p-value is calculated using an Unpaired t-test, which is a statistical test used to determine if there is a significant difference between the means of two independent groups. Most of the p-values are greater than 0.05, which suggests that there were no statistically significant differences between the placebo and carrageenan treatments for most of the endpoints. However, there were significant differences detected for intestinal permeability (lactulose-mannitol ratio) and zonulin. The footnote indicates that the p-values were calculated using unpaired t-tests comparing the within-subject differences between the two exposition sequences (Placebo-Carrageenan and Carrageenan-Placebo), also see methods.
Scientific Validity
  • Appropriateness of statistical methods and completeness of reporting: The presentation of results for all pre-specified study endpoints is a strength of the study, as it reduces the potential for selective reporting. The use of mean and standard error is appropriate for summarizing continuous data. The use of t-tests is also appropriate for comparing the means of two groups. However, it's important to consider the limitations of t-tests, particularly when the data are not normally distributed. It is unclear if the data met the requirements for the t-tests, and the authors should discuss this.
  • Consideration of effect sizes and multiple comparisons: The table provides a comprehensive overview of the study's findings. However, it would be helpful to include effect sizes to provide a better sense of the magnitude of the differences between the treatment groups. It would also be helpful to discuss the potential for multiple comparisons. The authors should also justify their choice of statistical test and discuss any potential limitations.
Communication
  • Clarity and organization of the table: The table is well-organized and presents a comprehensive overview of the study's findings for all pre-specified endpoints. The clear column headings and consistent formatting make it easy to compare the results for the placebo and carrageenan treatments. The inclusion of sample sizes (n1 and n2) is helpful for interpreting the results. The use of a footnote to explain the statistical method used (Unpaired t-test) is appropriate. However, the table could benefit from including confidence intervals for the mean differences to provide a better sense of the precision of the estimates.

Discussion

Key Aspects

Strengths

Suggestions for Improvement

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