Verified Rigor RecordRigor Reviewed

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Age Related Changes in NAD+ Metabolism Oxidative Stress and Sirt1 Activity in Wistar Rats · 10.1371/journal.pone.0019194

Name: Verified Rigor Record — Age Related Changes in NAD+ Metabolism Oxidative Stress and Sirt1 Activity in Wistar Rats · 10.1371/journal.pone.0019194
Item: Age Related Changes in NAD+ Metabolism Oxidative Stress and Sirt1 Activity in Wistar Rats · 10.1371/journal.pone.0019194
Rating: 71
Author: Αlpha¹

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Αlpha¹ Editorial

This rigor review was examined and confirmed by Αlpha¹ Editorial · July 6, 2026

Αlpha¹ Rigor Review

Rigor Reviewed

WARN

Warnings to address · Observational Study

Eight dimensions — click a segment for details

Critical

High-priority

Model-reported confidence: moderateReviewer agreement: lowEngine: 7.6.0Source: full text

Not verified in this report

References were not verified against Crossref/OpenAlex.
No reported statistical tests were found to recompute.
No data or code availability links were detected to verify.

This Αlpha¹ Rigor Review uses AI Rigor Reviewers trained on a curated corpus of high-fidelity and retracted papers, with expert supervision and curation. It can still make mistakes; verify each finding against the source before relying on it.

The paper has a clear scientific premise and reports detailed methods, funding, and limitations, but suffers from significant reporting gaps in study design (no power analysis, inclusion criteria, or controls), key resources (missing catalog/RRIDs), statistical analysis (no exact p-values or effect sizes), and data/code availability (no statement). Several copyedit errors ('brain' mislabeling) suggest inadequate proofreading.

All eight dimensions were applicable and scored. Two checklist sub-criteria (randomization method, randomization unit) were set to N/A as the study is observational. Reviewers diverged on study design (1 fail, 2 warn), biological variables (1 warn, 2 pass), ethical approvals (2 pass, 1 warn), key resources (1 fail, 1 pass, 1 warn), statistical analysis (1 fail, 2 warn), and reporting transparency (1 pass, 2 warn); the synthesized statuses reflect a weighted consensus prioritizing specific verifiable evidence.

12 major claims checked against the paper's own evidence: all adequately supported.

3 integrity concerns flagged (0 high).

Rigor

Eight Dimensions3 pass · 3 warn · 2 fail

12 major claims12 supported

The introduction reviews the free-radical theory of aging, the roles of PARP and Sirt1, and states that 'no study has yet reported on changes in NAD+ levels during the aging process' (Introduction). The rationale linking oxidative stress to NAD+ depletion and downstream effects is logically developed. Limitations of prior research are implicitly addressed by the novelty of the study.

Sub-criteria

prior work citedADEQUATE

premise rationaleADEQUATE

limitations addressedADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 95% conf

The paper establishes a strong scientific premise by citing extensive prior work on oxidative stress, PARP, and sirtuins, and clearly identifies a gap in knowledge regarding NAD+ changes during aging.

Evidence

direct quote[Introduction, first paragraphs]

“The 'free-radical theory of aging', initially proposed by Harman (1956) suggests that oxidative damage occurs with advanced aging...”

direct quote[Introduction, last paragraph]

“Though a number of studies have demonstrated elevated levels of oxidative stress associated damage in aged tissue, to our knowledge, no study has yet reported on changes in NAD+ levels during the aging process.”

direct quote[Introduction, last paragraph]

“Our results suggest that oxidative stress induced NAD+ depletion could play a significant role in the aging process.”

PASSReviewer 2· 95% conf

The paper establishes a clear scientific premise linking oxidative stress, NAD+ depletion, PARP activation, and Sirt1 activity in aging, citing relevant prior research.

Evidence

direct quote[Introduction, paragraphs 1-6]

“An important mechanism contributing to aging is oxidative stress. The "free-radical theory of aging", initially proposed by Harman (1956) suggests that oxidative damage occurs with advanced aging due to an imbalance between free radical and reactive species (ROS) production...”

direct quote[Introduction, last paragraph]

PASSReviewer 3· 90% conf

The study clearly establishes the free-radical theory of aging and prior work on oxidative stress, PARP, NAD+, and Sirt1, providing a logical rationale for the investigation.

Evidence

direct quote[Introduction, paragraphs 1-6]

“An important mechanism contributing to aging is oxidative stress. The 'free-radical theory of aging', initially proposed by Harman (1956) suggests that oxidative damage occurs with advanced aging...”

direct quote[Introduction, paragraphs 5-6]

paraphrase[Introduction, paragraph 6]

“The authors state that no study has reported on changes in NAD+ levels during aging, but they do not elaborate on design or methodological limitations of prior studies.”

This is an observational cross-sectional study comparing three age groups of rats (3, 12, 24 months). No randomization method or unit is reported, nor is any blinding of investigators to age group during assays. An a priori power analysis is absent. Inclusion/exclusion criteria are not stated. Outlier handling is not described. Positive and negative controls for the biochemical assays are not discussed (though β-actin is used as a loading control for western blots). Independent replication of findings is not explicitly reported or planned. The study uses n=8 rats per group, which is reasonable, but lack of pre-specification and blinding lowers confidence.

Sub-criteria

controlsINADEQUATE

power analysisNOT REPORTED

blinding levelsNOT REPORTED

outlier handlingNOT REPORTED

randomization unitNOT REPORTED

inclusion exclusionNOT REPORTED

randomization methodNOT REPORTED

replicate distinctionADEQUATE

independent replicationNOT REPORTED

Per reviewer (3) — the votes behind the verdict

FAILReviewer 1· 95% conf

The study lacks several key design elements: no power analysis, no inclusion/exclusion criteria, no outlier handling, no positive/negative controls, and no independent replication. Only biological/technical replicate distinction is adequately reported.

Evidence

direct quote[Data Analysis section]

“Results obtained are presented as the means ± the standard error of measurement (SEM) of at least eight animals per age group analysed in duplicate.”

absence[Methods]

No inclusion/exclusion criteria or outlier handling is reported.

paraphrase[Western Blots and Immunofluorescence methods]

“β-actin served as an internal control, and titration series and conjugate controls were performed for immunofluorescence. No explicit positive/negative assay controls are described.”

WARNReviewer 2· 85% conf

The study design is described, but critical elements like randomization, blinding, and pre-specified inclusion/exclusion criteria are not reported; the study is observational and lacks a pre-registered protocol.

Evidence

absence[Methods, Animals section]

No randomization method or unit is described.

absence[Methods]

Blinding is not mentioned.

direct quote[Methods, Data Analysis]

“Results obtained are presented as the means ± the standard error of measurement (SEM) of at least eight animals per age group analysed in duplicate.”

WARNReviewer 3· 85% conf

The study is observational, comparing age groups without randomization or blinding. Key design elements such as sample size justification, inclusion/exclusion criteria, and outlier handling are not reported.

Evidence

absence[Methods, Data Analysis]

No a priori power analysis or sample size justification is provided.

absence[Methods, Animals]

No inclusion/exclusion criteria are stated.

absence[Methods]

No blinding of investigators or assessors is mentioned.

The study uses female Wistar rats, with age groups specified (3, 12, 24 months). Sex is reported (female), and since only one sex is used, a scientific justification is needed but is absent; however, this is scored as not_reported. Species, strain, and source are reported. Housing conditions (light/dark cycle, temperature, ad libitum food and water) are described. Age and health status are given. Demographics (human) are not applicable.

Sub-criteria

demographicsN/A

sex reportedADEQUATE

sex justifiedNOT REPORTED

age weight healthADEQUATE

housing conditionsADEQUATE

species strain sourceADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 90% conf

Sex, age, and housing conditions are adequately reported, but weight/health status, source of animals, and justification for using only females are missing.

Evidence

direct quote[Animals section, Materials and Methods]

“Female wistar rats were used and experiments were designed to target the following age groups: 3 months old... 12 month old... 24 month old.”

direct quote[Animals section]

“The animals were individually housed in an environmentally controlled room under a 12∶12 hour light/dark cycle at 23°C and were given commercial rat chow and water ad libitum.”

absence[Animals section]

No justification for using only females, no weight or health status, and no animal source are provided.

PASSReviewer 2· 90% conf

Sex, age, weight, species/strain, and housing conditions are adequately reported, meeting all applicable criteria for this animal study.

Evidence

direct quote[Methods, Animals]

“Female wistar rats were used and experiments were designed to target the following age groups: 3 months old (equivalent to a young human adult aged 20 years), 12 month old (equivalent to a middle-aged human aged 40 years) and 24 month old (equivalent to an aged human greater than 80 years). The animals were individually housed in an environmentally controlled room under a 12∶12 hour light/dark cycle at 23°C and were given commercial rat chow and water ad libitum.”

PASSReviewer 3· 90% conf

Sex, age, species/strain, housing conditions are adequately reported. Health status is partially inferred from the age groups, and no explicit health assessment is provided.

Evidence

direct quote[Methods, Animals]

“Female wistar rats were used and experiments were designed to target the following age groups: 3 months old...12 month old...and 24 month old...”

absence[Methods, Animals]

Sex justification is not provided beyond the choice of female rats.

absence[Methods, Animals]

Health status is not explicitly reported beyond the age categories.

The paper states 'All experiments were performed according to the Animal Ethics Committee of the University of Sydney.' This names the approving body and indicates compliance. No protocol number is given, but the statement is clear and sufficient. Regulatory compliance is implied by following the institutional committee.

Sub-criteria

iacuc statementADEQUATE

informed consentN/A

irb ethics statementN/A

regulatory complianceADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 100% conf

The study reports approval by the Animal Ethics Committee of the University of Sydney, which is an adequate ethical statement for animal research.

Evidence

direct quote[Animals section, Materials and Methods]

“All experiments were performed according to the Animal Ethics Committee of the University of Sydney.”

WARNReviewer 2· 80% conf

An animal ethics committee is named, but no protocol number is provided; regulatory compliance is mentioned only implicitly, and the statement is incomplete by current standards.

Evidence

direct quote[Methods, Animals]

“All experiments were performed according to the Animal Ethics Committee of the University of Sydney.”

PASSReviewer 3· 90% conf

The study reports animal ethics committee approval from the University of Sydney, which meets the standard for animal research.

Evidence

direct quote[Methods, Animals]

“All experiments were performed according to the Animal Ethics Committee of the University of Sydney.”

absence[Methods, Animals]

No specific regulatory framework (e.g., Australian Code for the Care and Use of Animals) is cited.

The paper lists antibodies in Table 1 with source but no catalog numbers or RRIDs. Reagents in the 'Reagents and Chemicals' section are sourced from Sigma-Aldrich, Invitrogen, etc., but catalog numbers are omitted. The animal strain is stated as 'Wistar rats' but no vendor or breeder is given. Software (ImageJ) is mentioned without version. None of the applicable resources are fully identified.

Sub-criteria

mycoplasma testingN/A

reagents identifiedINADEQUATE

organisms identifiedINADEQUATE

antibodies identifiedINADEQUATE

cell line authenticationN/A

software tools identifiedINADEQUATE

Per reviewer (3) — the votes behind the verdict

FAILReviewer 1· 95% conf

Key resources are inadequately identified: antibodies lack catalog numbers, reagents lack catalog numbers, animal source is missing, and software version is not provided.

Evidence

paraphrase[Table 1]

“Table 1 provides antibody type, antigen, dilution, and source (e.g., Alexis Biochemicals) but not catalog numbers or RRIDs.”

direct quote[Reagents and Chemicals section]

“Phosphate buffer solution (PBS) was from Invitrogen (Melbourne, Australia). Nicotinamide, bicine, β-nicotinamide adenine dinucleotide reduced form (β-NADH)... were obtained from Sigma-Aldrich (Castle-Hill, Australia).”

absence[Animals section]

Animal source is not specified.

PASSReviewer 2· 90% conf

Key resources including antibodies, reagents, and software are largely identified with vendors and catalog numbers, though mycoplasma testing is not applicable and cell line authentication is not applicable.

Evidence

direct quote[Table 1]

“Antibody: (E)-4-Hydroxynonenal; Type: Polyclonal; Antigen: Free HNE (E)-4-hydroxynonenal; Dilution: 1:1000; Source: Alexis Biochemicals”

direct quote[Methods, Reagents and Chemicals]

“Nicotinamide, bicine, β-nicotinamide adenine dinucleotide reduced form (β-NADH), 3-[-4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)... were obtained from Sigma-Aldrich (Castle-Hill, Australia).”

WARNReviewer 3· 85% conf

Antibodies and reagents are identified with vendors and catalog numbers, but several important resources lack lot numbers or RRIDs. Mycoplasma testing and cell line authentication are not applicable.

Evidence

direct quote[Table 1]

“Table 1 List of primary antibodies used.”

direct quote[Methods, Western Blots]

“The bands were quantified by ImageJ software”

direct quote[Methods, Animals]

“Female wistar rats were used”

The paper uses one-way ANOVA with Tukey's post-hoc test. No verification of normality or equal variance is reported. Exact p-values are not given; only 'p<0.01' is reported throughout. Effect sizes and confidence intervals are not reported. The statistical software is not named beyond the microplate reader software. Data presentation includes bar graphs with SEM and individual n per group. Mathematical plausibility checks are not possible due to lack of raw data.

Sub-criteria

tests namedADEQUATE

exact p valuesINADEQUATE

effect sizes ciNOT REPORTED

data presentationADEQUATE

software identifiedNOT REPORTED

assumptions verifiedNOT REPORTED

mathematical plausibilityN/A

Per reviewer (3) — the votes behind the verdict

FAILReviewer 1· 95% conf

Statistical reporting is inadequate: exact p-values are not given, effect sizes and confidence intervals are absent, assumptions are not verified, and software is not identified. Only the test name is adequately reported.

Evidence

direct quote[Data Analysis section]

“One way analysis of variance (ANOVA) and post hoc Tukey's multiple comparison tests were used to determine statistical significance between treatment groups.”

direct quote[Results and figure legends]

“Significance * p<0.01 compared to 3 month old rats.”

absence[Data Analysis section and Results]

No effect sizes, confidence intervals, or software are reported.

WARNReviewer 2· 85% conf

Statistical tests are named (one-way ANOVA, Tukey's post-hoc), but assumptions are not verified, exact p-values are reported only as thresholds (p<0.01), effect sizes and confidence intervals are absent, and software version is not identified.

Evidence

direct quote[Methods, Data Analysis]

“One way analysis of variance (ANOVA) and post hoc Tukey's multiple comparison tests were used to determine statistical significance between treatment groups.”

direct quote[Results]

“Significance * p<0.01 compared to 3 month old rats.”

absence[Results, entire paper]

No effect sizes or confidence intervals are reported.

WARNReviewer 3· 80% conf

Statistical tests are named (one-way ANOVA with Tukey post-hoc) and exact p-values are reported as '<0.01', but effect sizes, confidence intervals, and test assumption verification are absent. Data presentation includes bar graphs with SEM, but individual data points are not shown.

Evidence

direct quote[Methods, Data Analysis]

“One way analysis of variance (ANOVA) and post hoc Tukey's multiple comparison tests were used to determine statistical significance between treatment groups.”

direct quote[Results e.g., Figure 1 legend]

“Significance * p<0.01 compared to 3 month old rats.”

direct quote[Figures 1-9]

“All values are means ± S.E from tissue obtained from eight different rats for each age group.”

The paper does not include any statement regarding availability of data or code. There is no mention of deposition in a public repository or provision of custom code. This is a significant gap in transparency.

Sub-criteria

code sharingNOT REPORTED

accession numbersN/A

repository depositNOT REPORTED

data availability statementNOT REPORTED

Per reviewer (3) — the votes behind the verdict

FAILReviewer 1· 100% conf

No data or code availability statement is provided, and no data repository deposits or code repositories are mentioned.

Evidence

absence[Whole paper]

No data availability statement is present.

absence[Whole paper]

No repository deposit or code sharing mentioned.

FAILReviewer 2· 95% conf

No data availability statement is provided, and no data have been deposited in any repository; code sharing is not applicable as no custom code is described.

Evidence

absence[Entire paper]

No data availability statement is present.

FAILReviewer 3· 95% conf

No data availability statement, repository deposit, accession numbers, or code sharing are provided. The paper is from 2011, before widespread mandates, but by current rigor standards this is a fail.

Evidence

absence[Full text]

No data availability statement is present anywhere in the paper.

absence[Full text]

No repository deposit or accession numbers are mentioned.

absence[Full text]

No code sharing is mentioned.

Methods are comprehensive, covering reagents, tissue processing, and assays. No reporting checklist (e.g., ARRIVE) is referenced. Limitations are discussed in the final paragraph, acknowledging the single-sex design and rodent-to-human extrapolation. Conclusions are largely proportional, but the final sentence suggests a causal role for NAD+ depletion that is correlational. Funding sources and competing interests are declared.

Sub-criteria

funding coiADEQUATE

trial registrationN/A

reporting guidelineNOT REPORTED

methods completenessADEQUATE

all outcomes reportedADEQUATE

limitations discussedADEQUATE

conclusions proportionalINADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 90% conf

The paper provides detailed methods, reports all measured outcomes, discusses limitations, draws proportional conclusions, and includes funding and competing interest statements. The only missing element is a reporting guideline checklist.

Evidence

paraphrase[Materials and Methods]

“All experimental procedures are described in subsections with specific reagents, protocols, and quantification methods.”

direct quote[Discussion, paragraph 4]

“Young and aged rat models are relatively homogenous in their gene expressions in the various organs. This may be in contrast to young adult and aged human populations which may demonstrate much greater heterogeneity.”

direct quote[End of paper]

“Funding: This work was supported by grants to Professor Tailoi Chan-Ling from the National Health and Medical Research Council of Australia... The funders had no role in study design... Competing Interests: The authors have declared that no competing interests exist.”

WARNReviewer 2· 80% conf

Methods are detailed, funding and competing interests are reported, and limitations are discussed, but no trial registration or reporting guideline is applicable, and not all pre-specified outcomes are clearly linked to a pre-registered analysis plan.

Evidence

direct quote[End of paper, before References]

“Competing Interests: The authors have declared that no competing interests exist. Funding: This work was supported by grants to Professor Tailoi Chan-Ling from the National Health and Medical Research Council of Australia...”

direct quote[Discussion, paragraph 3]

WARNReviewer 3· 85% conf

Methods are described in detail, but trial registration is not applicable, and no reporting guideline is referenced. Limitations are discussed but conclusions slightly overstate causal implications. Funding and competing interests are declared.

Evidence

direct quote[Methods section]

“Assay of o- and m- tyrosine for the Measurement of Protein Carbonyl Formation...The assay for o- and m- tyrosine was performed as previously described.”

absence[Full text]

No reporting guideline is referenced.

direct quote[End of manuscript]

“Competing Interests: The authors have declared that no competing interests exist.”

15 copyedit issues flagged: mostly consistency, typo, grammar.

Αlpha¹ Rigor Action ItemsFinal pass before journal submission

In this post-publication audit, the published paper has several reporting deficiencies that reduce confidence in the robustness of its findings. The missing data availability, lack of exact p-values and effect sizes, absent animal source and health metrics, and copyedit errors (repeated 'brain' mislabeling) warrant a reader's caution and could support a request for an erratum or independent re-analysis of the raw data. The core premise and methods are described, but the statistical and resource reporting gaps limit reproducibility.

1.
HIGHdata code
Add a data availability statement in a separate section before References, specifying that raw data are available on reasonable request or have been deposited in a repository (e.g., Figshare, Dryad) with a DOI.
The complete absence of any data availability statement is a major transparency gap that undermines reproducibility.
2.
HIGHstatistics
Replace threshold p-values (e.g., 'p<0.01') with exact p-values (e.g., 'p=0.003') throughout the Results and figure legends.
Exact p-values allow readers to assess the strength of evidence precisely rather than relying on arbitrary thresholds.
3.
HIGHreporting
Correct all figure captions that incorrectly refer to 'brain' when the study used liver, heart, kidney, and lung (Figures 1, 7, 8 and Results subheadings).
The repeated 'brain' mislabeling is a clear copy-paste error that misrepresents the study and could confuse readers.
4.
HIGHstatistics
Include a statement verifying ANOVA assumptions (normality, equal variance) and describe any corrective actions taken (e.g., transformation or non-parametric test) in the Data Analysis section.
Without assumption checks, the validity of the ANOVA and post-hoc results is uncertain.
5.
HIGHstatistics
Report effect sizes (e.g., Cohen's d, eta-squared) and 95% confidence intervals for all group comparisons in the Results.
Effect sizes and CIs provide information about the magnitude and precision of effects, which is essential for interpretation.
6.
HIGHreporting
Add a scientific justification for using only female rats in the Methods (Animals section).
Using only one sex without justification limits the generalizability of the findings and raises questions about potential sex-specific effects.
7.
HIGHreporting
Add a statement on outlier handling criteria and whether any data points were excluded from analysis in the Data Analysis section.
Missing outlier handling criteria leaves the analysis vulnerable to undisclosed data manipulation.
8.
HIGHreporting
Add a protocol number for the Animal Ethics Committee approval and cite the specific regulatory framework (e.g., Australian Code for the Care and Use of Animals).
A protocol number and regulatory framework citation provide traceability and confirm compliance with standards.
9.
MEDIUMreporting
Add catalog numbers or RRIDs for all antibodies listed in Table 1.
Missing catalog numbers/RRIDs hinders replication of antibody-based assays.
10.
MEDIUMreporting
Add catalog numbers for all key reagents (e.g., chemicals, kits) in the Reagents and Chemicals section.
Catalog numbers enable researchers to obtain identical reagents for independent replication.
11.
MEDIUMreporting
Specify the vendor or breeder from which the Wistar rats were obtained, and report animal weights at the time of sacrifice in the Animals section.
The source and health status (weight) of animals are essential for reproducibility and assessment of baseline health.
12.
MEDIUMreporting
Identify the statistical software (e.g., GraphPad Prism, SPSS) with version number used for all analyses in the Data Analysis section.
Knowing the software and version allows others to verify the analysis and is a standard reporting expectation.
13.
MEDIUMreporting
Mention whether blinding of the experimenter to age groups was performed during biochemical assays and data analysis, or justify why it was not necessary.
Blinding reduces bias; its absence should be justified or acknowledged.
14.
LOWcopyedit
Standardize the use of 'SEM' throughout figure legends (currently mixed 'S.E' and 'SEM').
Inconsistent terminology detracts from professional presentation.
15.
LOWcopyedit
Add commas in the title: change 'Age Related Changes' to 'Age-Related Changes' and add commas between 'Metabolism', 'Oxidative Stress', and 'Sirt1'.
Minor punctuation fixes improve readability and formatting consistency.

Full text (64,139 chars)Analyzed: Jul 6, 2026

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