Review for 10.1186/s12876-025-04040-5 (2025)

Title: Comparative Serum Imidazole Propionate Profiling in Colorectal Adenoma and Cancer
by UPLC-MS/MS
Version: 1 Date: 04 May 2025
Reviewer's report:
The revised manuscript titled “Comparative Serum Imidazole Propionate Profiling in Colorectal
Adenoma and Cancer by UPLC-MS/MS” presents several improvements over the original
submission. The authors addressed many statistical concerns, improved the structure of figures
and tables, and clarified some methodological points. However, multiple major scientific and
technical issues remain only partially resolved, and substantial concerns related to language,
grammar, and terminology persist throughout the text.
One of the primary unresolved issues relates to the poor interbatch precision at the lower limit
of quantification (LOQ). The reported coefficient of variation (23.16%) exceeds the commonly
accepted FDA threshold of 20%, even though the LOQ was set at 0.5 ng/mL. The authors
attempt to justify this by stating that all samples were above 1 ng/mL and that the standard
applies to exogenous compounds. This justification is inadequate. Regardless of the endogenous
nature of the analyte, precision below the LOQ remains a fundamental requirement for any
assay intended to distinguish early pathological changes, particularly in biomarker research.
No additional methodological adjustments, replicate analyses, or confirmatory validation were
performed to support the use of this threshold or to confirm reliability at the lower range.
There is also a lack of clarity regarding the specificity of ImP for colorectal cancer. The exclusion
of patients with diabetes, obesity, hypertension, and chronic kidney disease—conditions
already associated with elevated ImP—does reduce confounding; however, it simultaneously
prevents the manuscript from addressing whether elevated serum ImP is specific to colorectal
neoplasia or reflective of broader systemic metabolic disturbances. The authors acknowledge
this limitation but do not attempt any subgroup analysis, matched comparison, or literature-
supported discussion to quantify this overlap. As a result, the specificity of ImP as a cancer
biomarker remains speculative.
Another issue is the lack of anatomical or histological subgrouping of colorectal lesions. While
the authors mention that their grouping strategy is based on the adenoma–carcinoma sequence,
the omission of polyp location, subtype, or molecular classification (such as CIN, MSI, or
CIMP pathways) introduces a potential source of biological and statistical confounding. The
gut microbiota, ImP metabolism, and carcinogenesis mechanisms differ significantly between
the right and left colon. Without stratifying the sample by these variables, the findings lose
mechanistic depth and may conflate heterogeneous biological processes.
Furthermore, the manuscript does not provide evidence of internal reproducibility or control
for pre-analytical variables. No mention is made of operator variability, sample handling times,
or between-batch normalization strategies, which are essential components of robust biomarker
development. The response letter asserts that the team “optimized procedures” but offers no data
or supplementary documentation to support these claims. This undermines the reproducibility
and credibility of the analytical pipeline, particularly for clinical application.
The manuscript’s discussion of causality remains problematic. Although the authors
acknowledge the cross-sectional nature of the study, several sections continue to use causative
language such as “ImP may play a role” or “ImP contributes,” which implies a directionality
unsupported by the design. The absence of fecal or tissue ImP measurements further weakens the
ability to distinguish whether serum ImP levels reflect microbial production, tumor metabolism,
or systemic changes. This leaves the biological interpretation of elevated serum ImP incomplete
and speculative.
Language and grammar require further professional editing. Numerous syntactic and lexical
errors persist, including inconsistent verb tenses, awkward phrasings, and run-on sentences.
For instance, phrases such as “Our studys’s single-center design,” “IMPs’s biological value,”
or “we used internal standard can correct error of matrix” are grammatically incorrect or
awkwardly constructed. There is also inconsistent use of abbreviation formatting (e.g., “IMP,”
“ImP,” and “Imp” are used interchangeably), and the full name “imidazole propionate” is
sometimes introduced multiple times without consistency. Standard terminology from analytical
chemistry and molecular oncology should be used more precisely; for example, “selectivity” and
“specificity” are at times conflated, and “detection method” is used ambiguously when referring
to both assay validation and clinical application.
Figures and tables, while improved, still contain issues. Some chromatograms lack axis labels
and intensity scales. Statistical annotations on boxplots and ROC curves are inconsistently
formatted or inadequately described. Table 6 mixes parametric and non-parametric data in a
single layout without clear subgroup explanation, and confidence intervals are used without
explicit clarification as to whether they pertain to means or medians.
Finally, several limitations remain unacknowledged or insufficiently discussed. There is no
external validation cohort, no assessment of pre-analytical error sources, no evaluation of
batch-to-batch instrument performance, and no exploration of longitudinal predictiveness.
Additionally, the absence of microbiome sequencing or ImP quantification in feces or tumor
tissue weakens the biological plausibility of the ImP-CRC association and limits insight into the
metabolite’s origin and functional relevance.
While the revised manuscript demonstrates improvements in structure and partial responsiveness
to prior reviewer critiques, the remaining deficiencies in analytical validation, study design,
mechanistic insight, and language precision limit the manuscript’s scientific rigor. The data
suggest a potential association between serum ImP and colorectal neoplasia, but without stronger
validation and more rigorous reporting, the clinical relevance and biomarker value of ImP remain
unproven. Further refinement, including expanded cohort design, improved language quality,
and mechanistic experimentation, is necessary before the findings can be recommended for
publication in their current form.