Degradation of the Vaccine Additive Thimerosal by L-Glutathione and L-Cysteine at Physiological pH
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors“Suggestions and comments”
Dear authors, nowadays the articles are written to scientist that are involved in specific subject, even though they can be consulted by novels scientist or students, and the use of acronyms is widespread, but not always the significance is the same in all fields. That´s why I suggest specifying some of them that are in the text.
Pg 1. In the keywords RP-HPLC
Also mentioned flame absorption spectrometry (here it is preferable to add the acronyms FAAS, even though it is mentioned in the text in pg 4.
Pg 3, you describe, dI water, I suppose you mean deionized water..
What does it MCE membrane mean?
Pg 8: “after its incubation with RBC cytosol at 37° Celsius “- (what is RBC, may be “red blood cells”?) Please specify.
Figure 5.
The lines purple and green seem to have a mirror relationship. Do you have any explanation about this? It does any equilibrium exist between the species? While the orange line has an intermediate shape between the other lines. Do you perform any additional analysis to investigate the possibility of an equilibrium at pH 7.4 that may be present? Please comment.
In page 9. You express “ Based on these previous (correct, please, there is a mistake) studies obtained with RBC cytosol it appears that THI degrades within cells and the formed degradation product EtHg will then competitively (here is another mistake in typing) bind to biological thiols, such as GSH and/or Hb based on steric factors.”
From my point of view, more than steric factors that obviously could fit, it could be rationalized as an interaction of soft acid-soft base according to the nature of Hg and thiol compounds
Author Response
We greatly appreciate the constructive feedback on our manuscript by this reviewer and hereby respond to all questions as seen below:
Page 1: As requested we changed 'RP-HPLC' to 'reversed phase HPLC'. Although the reviewer suggested to change 'flame atomic absorption spectroscopy' to 'FAAS', we did not think this to be a good idea since some readers may not be familiar with this abbreviation that is widely used among analytical chemists that work in the area of atomic spectroscopy.
Page 3: We use the phrase 'dI water', which the reviewer wanted us to explain. We explain this abbreviation on page 4, line 6, which the reviewer may have missed.
The reviewer asks what 'MCE membrane' means. Since these letters are part of the product name of this Millipore product, we need to describe this product in detail in the experimental part just in case others want to reproduce our results.
Page 8: The reviewer had a question related to the sentence 'after its incubation with RBC cytosol at 37 Degree Celsius'. We respond that we explain the abbreviation 'RBC' in on the first page of the Introduction ('it means red blood cells').
The reviewer had a question related to Figure 5 and specifically the 'purple and green lines'. What can be seen in this figure are changes of the detected chromatographic Hg-peaks when increasing L-cysteine concentrations were added to the mobile phase. The green Hg-peaks refer to the result obtained with 10 mM Cys in the mobile phase, while the purple Hg-peaks refer to the results obtained with 20 mM Cys in the mobile phase. Accordingly, the green peaks correspond to an intermediary Hg-species with regard to the degradation of thimerosal, while the purple peaks correspond to the final Hg-degradation product (i.e. a EtHg-Cys adduct).
Page 9: We are grateful to the reviewer for spotting these typos and have corrected the latter to 'previous' and 'competitively'.
We agree with this reviewer that 'more than steric factors', namely an interaction of soft acid-soft base explains our results. We thank the reviewer again for the time to read our manuscript and to provide feedback that we hope to have addressed in sufficient detail.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript addresses the degradation of thimerosal by low-molecular-weight thiols — L-glutathione and L-cysteine — at physiological pH, using LC-FAAS, ESI-MS, and 199Hg NMR. The authors report the formation of GS–EtHg and a putative Cys–EtHg adduct, demonstrating rapid degradation under their test conditions. The following issues need to be resolved before the final recommendation for publication:
- Clarify that 30% ACN was used — this should not be buried in methods.
- The autism discussion should be limited to background, avoiding unnecessary controversy unless directly supported by new data.
- The NMR shift from −743 ppm to −740 ppm is small and could fall within instrumental error; the key evidence is the multiplet collapse. Provide spectral overlays and integration data, and discuss whether the broad singlet could arise from exchange processes or conformational heterogeneity.
- The manuscript could explore whether GS–EtHg adduct formation is irreversible or trans-thiolation with proteins might occur in vivo. Include a mechanistic scheme for possible subsequent fates of GS–EtHg (e.g., transport, excretion, protein binding).
- Indicate GSH/Cys concentration directly in figure legends.
- Typos: “confiremd” → “confirmed” , “shelflife” → “shelf life” , “revelaed” → “revealed” , “competitivelky” → “competitively” , “therei” → “therein”
Author Response
We thank the reviewer for the constructive feedback on our manuscript and will address these in detail point-by-point below:
Ad 1) We agree with this statement and have modified the abstract and the last line of the introduction to point out that our results were obtained in the presence of 30% of acetonitrile. We note that in Fig, 2, 5 and 6 the caption clearly states that our results were obtained in the presence of 30% of acetonitrile.
Ad 2) We are grateful for this comment and have actually prepared our manuscript with this idea in mind.
Ad 3) As requested we have provided a spectral overview (see pdf attachment), which reiterates our results depicted in Figure 6. Since these results just reiterate the results depicted in Figure 6 we have not seen a need to incorporate this figure into the revised manuscript. We have also integrated the data which provided results for thimerosal of 607,7740.99 area units and for thimerosal plus GSH of 450,193.80 area units. We attribute the lower value for the latter sample by the considerably more noisy background. All of our experiments were performed at room temperature. Based on the obtained data, we cannot distinguish whether the broad signal could arise from exchange processes or conformational heterogeneity. While low temperature experiments could be used to resolve this issue, we believe that this is beyond the overall scope of the manuscript.
Ad 4) The reviewer suggest to discuss whether the GS-EtHg adduct formation is irreversible or trans-thiolation with proteins might occur in vivo. While we greatly appreciate this suggestion, we think it is better to just report our results which were obtained in the presence of 30% acetonitrile, which is not physiological. Nevertheless, our results were obtained at pH 7.4.
Ad 5) The reviewer suggests to indicate the GSH/Cys concentration in the figure legends. We think that providing these concentrations as an inset in the corresponding figures is sufficiently clear from a readers point of view.
We thank the reviewer again for the valuable comments and hope that addressing them has improved the overall quality of the revised manuscript.
Author Response File: 
Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript deals with the bioinorganic chemistry and biodegradation of vaccine additive thiomersal. The amount of research presented is somewhat limited, nevertheless interesting and relevant.
Concerning the technical quality, I recommend a thorough spellcheck of the manuscript as it contains numerous errors. Also, Figure 6 is misspelled as 'Figure 6199'.
I would recommend to move Tables 1 + 2 to Supplementary Materials. It might be good to involve therein also the MS spectrum of the putative Cys-EtHg adduct (mentioned in the manuscript only as 'data not shown').
Comments on the Quality of English Language
The technical quality of the manuscript could be improved, as it contains numerous misspellings.
Author Response
We thank the reviewer for providing us with valuable feedback on our manuscript. Please find our point-by-point response to each of the comments/criticisms below.
Ad 1) We have addressed all typos in the revised manuscript. We think Figure 6 is not misspelled as 'Figure 6199', but rather that the term '199Hg-NMR' was too close to the 'Figure 6'.
Ad 2) The reviewer suggests to move Tables 1 and 2 to the supplementary material. We do not think that this is a good idea as the content of these tables is discussed in the manuscript. It would be onerous for the reader to always flip to the electronic supplementary information when reading the results and discussion of our manuscript.
Ad 3) The reviewer also suggested to add the ESI-MS spectrum of the EtHg-Cys adduct. Owing to the fact that GSH is much more abundant in cells than Cys we do not think that adding this spectrum is very important as it is rather similar to the GS-EtHg adduct depicted in Figure 3.
We thank the reviewer again for the valuable comments. Implementing them into the revised manuscript has hopefully improved its overall quality and clarity to be reconsidered for publication in Inorganics.
