Review for 10.1186/1471-230X-11-20 (2011)

Reviewer's report
Title:Acetaminophen-cysteine adducts during therapeutic dosing and following
overdose.
Version: 1Date:17 December 2010
Reviewer: Michael Mullins
Reviewer's report:
1. Is the question posed by the authors well defined? This is descriptive study to
explore the ranges of APAP-CYS concentrations in different dosing conditions
and among selected patients with overdose. The study aims to compare these
the concentrations found in these subjects and patients to previously suggested
threshold of 1.1 nmol/mL.
2. Are the methods appropriate and well described? Yes.
3. Are the data sound? Generally, but there are missing sequence numbers
among the overdose cohort. Also the acute overdose subset should be divided
further into acute overdose with and without hepatotoxicity.
4. Does the manuscript adhere to the relevant standards for reporting and data
deposition? Yes, except for the excluded patients in the overdose cohort.
5. Are the discussion and conclusions well balanced and adequately supported
by the data?
6. Are limitations of the work clearly stated? No. The authors opine that
APAP-CYS measurement is highly specific based upon 4 selected overdoses of
other medications for whom the history was known.
7. Do the authors clearly acknowledge any work upon which they are building,
both published and unpublished? Yes.
8. Do the title and abstract accurately convey what has been found? Yes.
9. Is the writing acceptable? Yes.
Please make your review as constructive and detailed as possible in your
comments so that authors have the opportunity to overcome any serious
deficiencies that you find and please also divide your comments into the following
categories:
• Discretionary Revisions (which are recommendations for improvement but
which the author can choose to ignore)
Please change occurrences of “where” used in place of the more correct “in
which”, unless describing position or location.
Please change occurrences of “levels” to “concentrations” when discussing a
measured or measurable quantity. It is acceptable to use brackets to indicate
concentration as in “[APAP-CYS]”.
Please use “patients” instead of “subjects” to refer to the people in the
observational series of overdoses.
• Minor Essential Revisions (such as missing labels on figures, or the wrong use
of a term, which the author can be trusted to correct)
Methods: Please change “clinical trials (n=3)” to “three clinical trials” so that “n”
only refers to numbers of subjects/patients.
Results / Observational study: “… and none of the patients who reported
ingesting hepatotoxins without acetaminophen had APAP-CYS detected.” Please
insert “four” before “patients”.
Results / Observational study: Please insert “selected” before the word “subjects”
in “Figures 3, 4, and 5 show the time course of … for subjects in the
acetaminophen overdose group…”
• Major Compulsory Revisions (which the author must respond to before a
decision on publication can be reached)
Background: The discussion which defines APAP-CYS should be clearer that
you are describing APAP adducts to proteins other than glutathione (the
tripeptide Glu-Cys-Gly) and other than N-acetylcysteine.
Methods: Observational study – The acute APAP overdose patients should be
further divided into those who actually had toxicity (elevated ALT or AST) and
those who had normal transaminases.
Also, the sequence numbers for the three hospitals in the accompanying table
are missing 15 patients. Please account for UHC 2, 4, 10, 12, and 13; SH 10 and
11; and D 3, 9-15, and 17.
APAP-CYS Protein Adduct Assay – Please clarify the importance of stating that
the assay has a lower limit of detection expressed in micromoles per liter of
assay solution and then stating that the results are expressed in nanomoles per
milliliter of serum. Does one milliliter of serum yield one milliliter of assay
solution?
Results / Clinical Trials: Please change “different from” to “lower than” in the
sentence starting with “The mean peak recorded adduct concentrations in Trial 2
were statistically different …”
Results: “Two placebo treated subjects in Trial 2 and 1 placebo treated subject in
Trial 3 had detectable APAP-CYS at the end of the study. All of these subjects
had detectable APAP-CYS levels [sic] at baseline …” First, what were their
starting APAP-CYS concentrations? Second, please justify why you did not
exclude these subjects from analysis. I would exclude these but retain them in
the discussion because the observation that they all had declining APAP-CYS
concentrations may generate new hypotheses.
Results / Observational study: “Thirty-five subjects [patients] were enrolled…”
The table of these patients includes three series of sequence numbers based
upon their hospital. However, there are 15 missing sequence numbers. Please
comment on why these patients were excluded.
Discussion: “The reasons for these differences …may have been related to
differences in assay storage or time intervals between sample collection and
analysis.” This is a highly speculative statement for which you provide no support
whatsoever. If the APAP-CYS concentrations change over time in serum
specimens “frozen at each institution and stored at -80 C until analysis” , then
you should either have assayed all specimens at the same time interval from
collection. While this may not have been practical, you could easily split samples
from the same patient/subject and assay the different aliquots of the same
specimen after different storage intervals to see whether the APAP-CYS
concentrations are truly unstable at – 80 Celsius (-112 Fahrenheit).
Although this statement is overly speculative, you could at least attempt to justify
it by indicating the mean and median duration of specimen storage among the
three trial groups.
“Slight differences in the sampling scheme among these three studies make
direct comparisons difficult. It is possible that these differences are due to
variations in sample timing, handling or processing or non-compliance with study
medication. “ Most of this statement appears to repeat the idea stated two
paragraphs earlier (see above) again without justifying or supporting it by
reporting the variations in sample timing, handling or processing”.
Sensitivity and specificity: Although it is plausible that APAP-CYS is specific for
APAP exposure, this study was not designed to determine sensitivity and
specificity.
Assuming for the sake of argument that you could meaningfully calculate
sensitivity and specificity from these data, I do not understand how you reached
a sensitivity of 97%. If sensitivity = true positives / (false negatives + true
positives) = true positives / all positives, then your sensitivity = 7/8 or 87.5%.
“At this time we cannot make conclusions regarding the lack of correlation
between APAP-CYS and ALT. The correlation between APAP-CYS and ALT
observed in the overdose cohort was statistically significant and similar to other
reports.” The first sentence appears to contradict the second sentence and the
last sentence in the Results section.
“Importantly, the highest levels [concentrations] of APAP adducts measured in
patients were in those with severe cases of APAP overdose who did not receive
timely treatment with NAC.” This statement is FALSE, unless you arbitrarily
redefine “timely treatment”. The highest [APAP-CYS] occurred in Patient SH-04
who received NAC at 7 hours with [APAP-CYS] of about 28 nmol/mL on the
graph.
Conclusion: I would agree that APAP-CYS may be useful in determining the
likelihood of APAP overdose in late presenting patients who do not reviely their
APAP intake.
Table 2: Pt UCH-06 does not have the peak ALT reported here. Also, only 2
acute overdose patients out of the 12 patients ever had an [ALT] above treated
within 3 hours 2 x the ULN.
Given that APAP-CYS correlates with ALT, it appears that the primary situation in
which APAP-CYS would be a useful biomarker would be that in which a
late-presenting patient either who either denies taking APAP or is unable to give
history when presenting with transaminase elevations with concern for APAP
overdose. It would be useful in refining the case definition for acute liver failure
resulting from occult APAP overdose.
Supplementary table: Please change “subject number” to “patient number”
Patient UCH-06 has a missing value for the peak ALT concentration.
Patient UCH-09 with 21 grams over 5 days is just 1 gram away from the trial 3
subjects who all took 20 grams over 5 days. The APAP-CYS and ALT
concentrations are similar to those found inTrial 3.
Although not discussed explicitly in this paper, one wonders whether
[APAP-CYS] is proportional to the ingested dose of APAP, the highest observed
[APAP], and/or the time interval until treatment with NAC.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.