Impact of Vitamin A Supplementation on Immune System in Multiple Sclerosis Patients: Complete Statistical Analysis

1. Trial at a Glance

This page covers the registry results for Impact of Vitamin A Supplementation on Immune System in Multiple Sclerosis Patients (NCT01225289), a randomized phase 4 study. The primary outcome is Difference Serum Levels of High-sensitive C-reactive Protein (Hs-CRP), Before and After of Supplementation.

2. Clinical Question

The registry describes a comparison of supplementation and placebo on the immune system and Th1/Th2 balance. The listed primary outcome is specifically the difference in serum Hs-CRP levels before and after supplementation, measured in mg/L. The secondary outcomes include a measure of IL-4 in stimulated peripheral blood mononucleated cells, the retinol-binding-protein-to-transthyretin ratio, and a cell proliferation assay.

The question is about differences in measured outcomes between assigned groups; it is not a direct test of clinical symptoms, disability, relapse frequency, or long-term disease course. Those outcomes are not listed in the registry record. The record also does not report a detailed definition of the analysis population or whether all randomized participants contributed data to each outcome.

3. Trial Design

This was a randomized, quadruple-masked, parallel-group phase 4 trial sponsored by Tehran University of Medical Sciences. It enrolled patients with relapsing-remitting multiple sclerosis and compared vitamin A with placebo. The registry lists 36 participants as the enrollment, and the reported arm summaries contain 18 participants for vitamin A and 17 for placebo. How those arm counts relate to the full enrollment, including any withdrawals or exclusions, is not reported in the registry record.

The vitamin A group received 25000 IU per day, while the placebo group received one capsule per day. The planned supplementation period in the trial description was 6 months. The primary outcome was the difference in serum Hs-CRP levels between the first day and after 6 months. The registry also lists three secondary outcomes: IL-4 levels in stimulated peripheral blood mononucleated cells, the retinol-binding-protein-to-transthyretin ratio, and peripheral blood mononucleated cell proliferation measured by a BrdU colorimetric assay.

The registry dates the study start to 2009-10 and primary completion to 2013-12, and lists the study status as completed. Randomization and masking are reported, but the method used to generate or conceal the allocation sequence, and the practical details of masking, are not reported in the registry record. The planned sample-size justification, the trial's original statistical analysis plan, and details of any interim analyses are also not reported. The registry record identifies a Wilcoxon (Mann-Whitney) analysis for the primary comparison, but does not provide a fuller account of the prespecified analysis procedures.

4. Participant Flow and Accounting

The registry reports how participants moved through each stage of the study. All counts below are exactly as posted; missing counts are not inferred from other stages.

Overall Study

5. Baseline Characteristics

The table reproduces the baseline characteristics posted in the registry, which describe the participants analysed at baseline and help show whether the randomized groups were comparable.

6. Primary Endpoint

The registry lists the primary outcome as Difference Serum Levels of High-sensitive C-reactive Protein (Hs-CRP), Before and After of Supplementation, measured over first day and after 6 month.

The primary endpoint is the difference in serum levels of high-sensitivity C-reactive protein (Hs-CRP) between the first day and after 6 months of supplementation. Hs-CRP is a blood measure used as an indicator of inflammation. In this trial it serves as a laboratory measure relevant to the stated interest in immune-system effects; it is not itself a measure of multiple-sclerosis symptoms or disease progression.

The registry describes the endpoint as continuous, reports values in mg/L, and summarizes the arm results using means and standard errors. A before-and-after outcome can capture change over the supplementation period, while a between-group comparison asks whether the observed change summaries differ between the randomized groups. The posted summaries give a mean of 1.2 for the vitamin A group and -0.6 for the placebo group; the recalculated unadjusted mean difference is 1.8.

The registry does not explain the biological rationale for selecting Hs-CRP as the primary measure, specify whether the endpoint was calculated as a particular signed change, or give a clinical threshold for interpreting its size. Thus the numerical result describes the posted laboratory outcome, not the clinical importance of that difference.

7. Results Posted in the Registry

• With Multiple Sclerosis/ Vitamin A: mean 1.20 versus -0.60 on control; difference 1.80 (95% CI 0.55 to 3.05), standardized effect d = 0.94; z-test p = 0.005.

8. Secondary Efficacy Endpoints

The registry also posts results for secondary outcomes. For each outcome below, the endpoint type, the test applied to the posted summary statistics, the result and its interpretation are given in turn. Secondary endpoints are usually not adjusted for multiple testing, so they support rather than confirm the primary finding.

Difference of IL-4 Levels in Supernatant of Peripheral Blood Mononucleated Cells (PBMCs) Stimulated With Phytohemagglutinin (PHA), Before and After of Supplementation

• Endpoint type: Continuous: a mean value per participant, compared as a difference in means.
• Test: Unadjusted two-sample z-test for the difference in means, using the posted standard errors.
• Result:
  • With Multiple Sclerosis/ Vitamin A: mean 2.50 versus 1.50 on control; difference 1.00 (95% CI -2.06 to 4.06), standardized effect d = 0.22; z-test p = 0.522.
• Interpretation:
  • The 95% confidence interval includes no difference, so these data do not show a statistically significant difference between With Multiple Sclerosis/ Vitamin A and the control. The interval gives the range of effects that remain compatible with the data.

Difference of Retinol Binding Protein (RBP) / Transthyretin (TTR) Ratio, (Difference of RBP/ TTR Ratio), Before and After of Supplementation

• Endpoint type: Continuous: a mean value per participant, compared as a difference in means.
• Test: Unadjusted two-sample z-test for the difference in means, using the posted standard errors.
• Result:
  • With Multiple Sclerosis/ Vitamin A: mean 0.20 versus -0.40 on control; difference 0.60 (95% CI -0.46 to 1.66), standardized effect d = 0.38; z-test p = 0.265.
• Interpretation:
  • The 95% confidence interval includes no difference, so these data do not show a statistically significant difference between With Multiple Sclerosis/ Vitamin A and the control. The interval gives the range of effects that remain compatible with the data.

Peripheral Blood Mononucleated Cells (PBMCs) Proliferation Assay (BrdU Colorimetric)

• Endpoint type: Continuous: a mean value per participant, compared as a difference in means.
• Test: Unadjusted two-sample z-test for the difference in means, using the posted standard errors.
• Result:
  • With Multiple Sclerosis/ Vitamin A: mean -0.06 versus -0.13 on control; difference 0.07 (95% CI -0.17 to 0.31), standardized effect d = 0.20; z-test p = 0.561.
• Interpretation:
  • The 95% confidence interval includes no difference, so these data do not show a statistically significant difference between With Multiple Sclerosis/ Vitamin A and the control. The interval gives the range of effects that remain compatible with the data.

Across the three secondary outcomes, the recalculated group comparisons do not show the same precision pattern as the primary outcome: each confidence interval includes zero. The registry does not list sponsor-reported inferential analyses for these secondary endpoints. Their posted means and standard errors permit descriptive comparisons, but do not by themselves establish a clear between-group difference.

The secondary measures address different laboratory features of immune response: IL-4 in stimulated cells, the retinol-binding-protein-to-transthyretin ratio, and cell proliferation. They are related to the trial's broader interest in immune-system effects, but they are not interchangeable with the primary Hs-CRP measure. The registry does not report a combined analysis or a formal test linking the secondary results to the primary outcome.

These results should carry less weight than a prespecified primary analysis. Several outcomes create multiple opportunities to observe an apparently notable result by chance, and a multiplicity adjustment or hierarchy among the secondary endpoints is not reported in the registry record. The results can therefore be viewed as additional descriptive evidence, not independent confirmation of the primary comparison. The registry also does not report enough information about analysis populations or missing observations to assess whether each secondary result was based on the same participants.

Secondary outcomes registered for this trial, for reference (only those above were recalculated):

• Difference of IL-4 Levels in Supernatant of Peripheral Blood Mononucleated Cells (PBMCs) Stimulated With Phytohemagglutinin (PHA), Before and After of Supplementation
• Difference of Retinol Binding Protein (RBP) / Transthyretin (TTR) Ratio, (Difference of RBP/ TTR Ratio), Before and After of Supplementation
• Peripheral Blood Mononucleated Cells (PBMCs) Proliferation Assay (BrdU Colorimetric)

9. Statistical Interpretation

10. Statistical Methods Explained

The registry reports a Wilcoxon (Mann-Whitney) analysis for the primary comparison. The posted arm summaries also permit a simple unadjusted recalculation of the difference between group means and an associated confidence interval. These methods answer related but not identical questions, and the distinction matters when reading the results.

What does the reported rank-based test compare?

The Wilcoxon (Mann-Whitney) test is a rank-based method for comparing two independent groups. Rather than relying directly on a difference between means, it uses the ordering of observations across groups. It can be useful when a comparison is framed in terms of distributions or when the assumptions of a mean-based model are a concern. Its interpretation is not automatically a test of medians: a location-shift interpretation requires distributional conditions that are not described in the registry record.

The registry reports p <0.01 for this test and identifies a 95% confidence level, but the confidence-interval limits and test statistic are not reported in the registry record. The precise analysis details beyond the named method are also not reported.

What does the recalculated mean difference represent?

The recalculated estimate compares the posted mean change summaries for the two groups. A positive difference means the vitamin A group's reported mean change summary is higher than the placebo group's. Its 95% confidence interval describes a range of values compatible with the data and the assumptions used in that recalculation. It is not a guarantee that the true difference lies within the interval, and it does not account for sources of uncertainty that are not represented in the posted summary statistics.

The recalculation is unadjusted and uses the posted means and standard errors. It is not the same as the registry's rank-based test, and the registry does not report that the sponsor used this mean-based procedure. Since the discrepancy flag is false, no discrepancy between the sponsor's analysis and the recalculation is identified in the registry record. That does not make the methods interchangeable.

How should standard errors be understood?

The arm summaries report standard errors, which describe uncertainty in an estimated mean. They are not the same as standard deviations, which describe variation among individual observations. Neither standard errors nor group means show the shape of the underlying data, the presence of outliers, or whether the distribution differs between groups. Those features cannot be assessed from the posted summaries alone.

In general, a confidence interval combines an estimate with its uncertainty under a particular statistical method and assumptions. In general, greater dispersion or fewer observations tends to produce less precise estimates. Over-dispersion, meaning more variability than an assumed model allows, can make model-based uncertainty too small if the model does not account for it. The registry record does not provide individual-level data or enough diagnostic information to evaluate distributional assumptions for this trial.

What do the planning illustrations assume?

The sample-size planning illustrations use a two-sided significance level of 0.05, equal allocation between the two arms, and a normal approximation. They consider effects equal to 100%, 75%, or 50% of the observed effect. The planning assumptions are not reported as the original trial's design assumptions; the trial's own sample-size justification and design assumptions are not reported in the registry record.

These calculations are useful as illustrations of how estimated sample needs change when the target effect changes under those assumptions. They do not replace the trial's original design rationale, which is unavailable, and they do not establish that the trial was designed around the observed results.

What remains unclear about the analysis?

The registry record does not report the original statistical analysis plan, how the analysis population was defined, how missing outcome measurements were handled, or whether any covariate adjustment was planned. It also does not describe interim analyses. Such details can affect interpretation, particularly when several outcomes are reported, and cannot be reconstructed from the group summaries alone.

11. Confidence Intervals and Precision

The recalculated primary mean difference is 1.8 mg/L, with a 95% confidence interval from 0.545 to 3.055. This interval gives a sense of the precision of the unadjusted mean comparison under its calculation assumptions. The range is entirely above zero, but it still spans a range of possible magnitudes. The interval does not show which value is clinically important, because a threshold for clinical importance is not reported in the registry record.

The registry's own primary analysis reports a 95% confidence level, but its interval limits are not reported in the registry record. Its Wilcoxon (Mann-Whitney) test result, p <0.01, should not be paired with the recalculated interval as though both came from the same procedure. The two results arise from different methods.

For the secondary outcomes, the recalculated confidence intervals include zero. This means those summaries are compatible with both a difference in either direction and no difference under the recalculation assumptions. The registry does not report sponsor confidence-interval bounds for those outcomes.

Confidence intervals describe statistical uncertainty, not the range of individual participants' results or the probability that a particular effect is true. The available summaries also do not show the distribution of measurements, participant-level variability, or how missing observations may have affected precision. These limitations matter when judging how broadly the estimates can be applied.

12. Planning a Similar Trial: Sample Size

This is a planning illustration, not a re-evaluation of the original trial. It asks: if a new study wanted to detect a difference like the one observed, or a smaller one, how large would it need to be? It is deliberately not “observed power” of the finished trial, which is not a meaningful quantity.

The planning figures are participants per arm, not total events. They are illustrations based on the observed effect and are not the original trial's design justification. The stated planning assumptions are a two-sided significance level of 0.05, equal allocation between the two arms, and a normal approximation. The target effect is set in turn to 100%, 75%, or 50% of the observed effect.

The 80% power column gives the number of participants per arm that the planning calculation estimates would be needed to have an 80% chance of detecting the specified effect under those assumptions, if that effect were the true effect and the calculation's model applied. The 90% power column gives the corresponding planning count for a 90% chance. These are design-planning concepts, not guarantees about what a future study would find.

Smaller target effects require more participants in these illustrations: detecting a smaller fraction of the observed effect entails a larger planned sample. The displayed counts vary by endpoint because the calculations use each endpoint's observed effect and variability. They should not be interpreted as evidence that the completed trial had a particular probability of detecting its results. This is not observed power.

The registry does not report the trial's original sample-size assumptions or justification, so the planning illustrations cannot establish whether the enrollment of 36 was chosen to detect a particular clinically important effect. They are best read as conditional examples of how sample requirements change when the target effect and stated assumptions change.

13. How the Sample Size Is Derived

Power is the probability that a study of a given size rejects the null hypothesis when a specific true effect exists. It depends on four things: the effect to detect, the variability of the outcome, the significance level (here a two-sided α of 0.05, so z_1−α/2 = 1.960) and the desired power (z_1−β = 0.842 for 80% and 1.282 for 90%).

Formula

n is the number of participants in each arm, Δ is the difference in means to detect and σ is the common standard deviation.

Worked calculation for this trial

1. Observed difference Δ = 1.800; pooled standard deviation σ = 1.907.
2. n = 2 × (1.96 + 0.842)² × 1.907² / 1.800² = 17.6, rounded up to 18 participants per arm for 80% power.

The smaller-effect rows in the table above repeat the same calculation with the effect multiplied by 0.75 and 0.5 (for ratios, applied to the logarithm of the ratio).

R code

alpha <- 0.05
z_a   <- qnorm(1 - alpha / 2)          # two-sided
delta <- 1.8                      # observed difference in means
sigma <- 1.90708408230201                     # pooled standard deviation
n_per_arm <- function(power, frac = 1) {
  ceiling(round(2 * (z_a + qnorm(power))^2 * sigma^2 / (delta * frac)^2, 6))
}
for (f in c(1, 0.75, 0.5))
  cat(sprintf("%3.0f%% of observed effect: n per arm = %s (80%% power), %s (90%% power)\n", 100 * f,
              format(n_per_arm(0.80, f), big.mark = ","), format(n_per_arm(0.90, f), big.mark = ",")))

# Check against R's built-in function (t-based, so very slightly larger)
cat("power.t.test:", round(power.t.test(delta = delta, sd = sigma, power = 0.80)$n, 1), "\n")

# Simulation: exact two-sample t-test using simulated means and variances
set.seed(1225289)
n    <- n_per_arm(0.80)
reps <- 20000
m_diff <- rnorm(reps, delta, sigma * sqrt(2 / n))             # difference in sample means
s2     <- sigma^2 * rchisq(reps, 2 * (n - 1)) / (2 * (n - 1))   # pooled sample variance
tstat  <- m_diff / sqrt(s2 * 2 / n)
cat(sprintf("Simulated power at n = %s per arm: %.3f (%d replicates)\n", format(n, big.mark = ","), mean(abs(tstat) > qt(0.975, 2 * (n - 1))), reps))

Output from running the code

100% of observed effect: n per arm = 18 (80% power), 24 (90% power)
 75% of observed effect: n per arm = 32 (80% power), 42 (90% power)
 50% of observed effect: n per arm = 71 (80% power), 95 (90% power)
power.t.test: 18.6 
Simulated power at n = 18 per arm: 0.784 (20000 replicates)

The simulation draws many sets of group means and variances at the planned sample size and counts how often the two-sample t-test rejects the null. It should land close to the target power; at very small required sample sizes it can fall below it, because the normal approximation is rough there.

14. Analyses Reported by the Sponsor

The registry reports the sponsor's primary comparison as a Wilcoxon (Mann-Whitney) test with p <0.01 and a 95% confidence level. The confidence-interval bounds and test statistic are not reported in the registry record. The reported test is the registry's own analysis and uses a rank-based method.

The recalculated mean difference is 1.8 mg/L, with a 95% confidence interval from 0.545 to 3.055 and an unadjusted p value of 0.005. This recalculation uses the posted group means and standard errors; it is not a reproduction of the sponsor's rank-based test. The two p-value statements are expressed at different levels of precision, and the methods differ, so the numerical results should not be treated as identical estimates.

The registry record does not flag a discrepancy between the posted result and the recalculation. The recalculation is therefore a complementary summary based on the posted statistics, while the sponsor's named method remains the reported analysis. Further details needed to compare the analyses, including individual observations and the sponsor's test statistic, are not reported in the registry record.

15. Multiplicity, Missing Data and Analysis Populations

The registry lists one primary outcome and three secondary outcomes. It does not report a multiplicity strategy, such as adjusting inference across outcomes or specifying a formal order in which outcomes would be tested. Without that information, unadjusted secondary comparisons should be interpreted cautiously: examining multiple outcomes increases the chance that at least one result may appear notable even when there is no underlying difference.

The primary analysis is identified as a Wilcoxon (Mann-Whitney) test, but the full statistical analysis plan is not reported in the registry record. Details such as the prespecified significance threshold for the primary test, handling of covariates, planned sensitivity analyses, or rules for selecting the analysis population are not reported. The significance level of 0.05 appears in the planning illustration assumptions, but that does not establish the original trial's inferential threshold.

Missing-data handling and participant flow are also not reported. Although enrollment is listed as 36, the primary arm summaries contain 18 and 17 participants, and the registry does not explain the accounting for the remaining enrollment or whether any participants lacked particular outcomes. The record does not say whether analyses followed assignment regardless of adherence, used only participants with observed outcomes, or applied any imputation method.

The registry does not report interim analyses. It also does not specify whether the posted outcome analyses were prespecified or whether any outcomes were changed during the study. These omissions do not demonstrate that particular practices occurred; they mean that the relevant safeguards and decisions cannot be assessed from the registry record.

16. Safety Results

The registry reports serious adverse events as affected participants divided by participants at risk.

Other (non-serious) adverse events

Participants with at least one non-serious event at or above the registry's 0% reporting threshold.

Most frequent non-serious adverse event terms

The registry reports zero serious adverse events among 18 participants at risk in the vitamin A group and zero among 17 at risk in the placebo group. These are the posted serious-event counts during the recorded safety period. They should be read as counts in the reported groups, not as proof that no serious events could occur or that the groups have equivalent safety.

For other adverse events, the registry reports zero among 18 participants at risk in the vitamin A group and one among 17 at risk in the placebo group. The registry does not provide a term for that event in the posted summary. Death counts are not reported in the registry record.

The small group counts and the absence of further event descriptions limit what can be inferred from these figures. The record does not explain event definitions, severity grading beyond the serious-event category, the period of observation, or how events were collected. These counts describe the registry's posted safety data; they do not support a broad comparison of risk.

17. What the Result Does, and Does Not, Mean

The primary result describes a difference in the mean change summaries for serum Hs-CRP between the vitamin A and placebo groups over 6 months. The recalculated mean difference is 1.8 mg/L, and its 95% confidence interval extends from 0.545 to 3.055. The registry's own rank-based analysis reports p <0.01. Together, these results describe a statistical contrast in the recorded laboratory measure, using two different methods.

They do not establish the clinical importance of the contrast. The registry does not give a threshold at which a change in Hs-CRP would be meaningful to patients with relapsing-remitting multiple sclerosis. It also does not report clinical outcomes such as symptoms, disability, relapse frequency, or long-term progression. A laboratory difference alone cannot answer whether participants experienced a meaningful clinical change.

The results do not show why the groups differed or establish that the difference was caused by a particular biological mechanism. Randomization is a design feature intended to support fair group comparisons, but the available registry summary does not provide enough detail to evaluate allocation procedures, adherence, missing data, or all other potential sources of bias.

The secondary outcomes do not supply clear statistical confirmation: their recalculated intervals include zero, and sponsor analyses for them are not reported in the registry record. The absence of a reported difference is not proof that the groups were identical; the available estimates may be imprecise, and the record does not report a formal equivalence or non-inferiority design.

Overall, the registry supports a description of the posted laboratory comparisons, not a broad conclusion about clinical benefit, lack of benefit, or safety. Those broader questions require outcomes and reporting details that are not included in the record.

18. Why This Trial Matters Statistically

This trial is statistically interesting because its primary outcome is a before-and-after laboratory measure compared between randomized groups, while the registry's reported analysis is rank-based and the posted group summaries also permit a mean-based recalculation. Seeing both makes clear why an estimate and a test result should be read in the context of the method that produced them.

The primary recalculated confidence interval excludes zero, whereas the secondary recalculated intervals include zero. This illustrates the distinction between a more precisely estimated primary comparison and less conclusive secondary comparisons. It also shows why secondary measures do not automatically provide confirmation of a primary finding, particularly when multiplicity handling is not reported.

The planning illustrations make another general point: sample requirements depend on the target effect and the assumptions used to calculate them. The illustrations are not evidence of the original design rationale, which is not reported in the registry record, and they should not be mistaken for observed power.

Finally, the record demonstrates how missing methodological details limit appraisal. Randomization and quadruple masking are reported, but the allocation process, participant flow, missing-data approach, analysis population, and full analysis plan are not. Those details are important for understanding the reliability and scope of trial estimates, even when outcome statistics are available.

19. Limitations

• The posted summary statistics are the only data these results rest on; patient-level data, full protocols and statistical analysis plans are not available.
• Recalculated results are simple, unadjusted calculations from posted summaries. They do not replace the sponsor's prespecified analysis.

The registry record provides limited detail about trial conduct and analysis. Although randomization and quadruple masking are reported, how the allocation sequence was generated and concealed, who was masked, and how masking was maintained are not reported. Adherence to the assigned supplement or placebo is also not described.

Enrollment is listed as 36, while the posted primary arm summaries report 18 and 17 participants. The record does not provide a participant flow account explaining this difference, nor does it say whether the same participants contributed to each outcome. The analysis population, exclusions, withdrawals, and missing-data methods are not reported.

The primary outcome is described as a before-and-after difference, but the record does not give a detailed formula or clarify the direction used to calculate change. Individual-level data, outcome distributions, and diagnostic checks are unavailable, limiting assessment of assumptions underlying the recalculated mean comparison. The sponsor's reported rank-based analysis is identified, but its test statistic and confidence-interval bounds are not reported.

The trial's original sample-size justification, statistical analysis plan, multiplicity strategy, and interim-analysis procedures are not reported in the registry record. The clinical importance of the Hs-CRP result is also not defined, and clinical outcomes such as symptoms, disability, relapses, or long-term progression are not listed. These limitations restrict both statistical appraisal and the range of conclusions that can be drawn from the posted laboratory results.

20. Related Tutorials

21. Related Statistical Calculators

22. Sources

• ClinicalTrials.gov: Impact of Vitamin A Supplementation on Immune System in Multiple Sclerosis Patients — NCT01225289.
• Registry record last updated 2014-03-14; status last verified 2014-02.
• Results first posted 2014-03-14.
• Data retrieved from ClinicalTrials.gov on 2026-11-09.
• Group sizes, means, event counts and sponsor-reported p-values are reproduced as posted. Effect estimates, confidence intervals and p-values not attributed to the sponsor are independently calculated from the posted summary statistics.

23. Record Summary

NCT01225289 is a completed, randomized, quadruple-masked, parallel-group phase 4 trial sponsored by Tehran University of Medical Sciences. It enrolled patients with relapsing-remitting multiple sclerosis and compared 25000 IU per day of vitamin A with one placebo capsule per day for 6 months. The registry lists enrollment of 36 and reports primary arm summaries of 18 and 17 participants.

The primary outcome was the change in serum Hs-CRP between the first day and after 6 months. The registry reports a Wilcoxon (Mann-Whitney) result of p <0.01. The unadjusted recalculated mean difference is 1.8 mg/L, with a 95% confidence interval from 0.545 to 3.055. Three secondary laboratory outcomes are also posted, but sponsor analyses for those outcomes are not reported. Important details about participant flow, missing data, multiplicity, and the original statistical plan are not reported in the registry record.