New Approaches to Determining the D/H Ratio in Aqueous Media Based on Diffuse Laser Light Scattering for Promising Application in Deuterium-Depleted Water Analysis in Antitumor Therapy

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presented me to review process focus on potential application of stable hydrogen isotopes for adjuvant therapy. In my humble opinion, the article could be considered for publication after substantial revisions. In particular:

1.       Please modify the title to better match the abstract and conclusions. It is currently a bit inconsistent. In my opinion, "Advances in the Development" can also be removed.

2.       The abstract definitely needs to be shortened. Please limit yourself to your own research. Please move information that is important for the manuscript to the introduction section.

3.       Please limit excessive linking of literature, for example [21-29]. Instead, please provide some examples (line 119-120).

4.       To enhance the manuscript, theoretical analysis regarding the stability of the structures for tested water samples in experimental condition is welcome.

5.       It is recommended to create a supporting information file containing the original spectra and visualization of the analyses performed in order to validate the conducted research.

6.       Due to the nature of the study, he is considering introducing a future prospective section.

7.       Instead of creating an Abbreviation section, please place appropriate abbreviations in the text where they first appear.

Author Response

Response to Reviewer 1 Comments

The authors thank the respected Reviewer for valuable comments aimed at eliminating erroneous conclusions, inaccuracies in wording and at improving the quality of the material of the article, in general, to meet the high requirements of Scientia Pharmaceutica.

Comments 1: Please modify the title to better match the abstract and conclusions. It is currently a bit inconsistent. In my opinion, "Advances in the Development" can also be removed.

Response 1: The authors agree with the opinion of the respected, thank you very much! The title of the manuscript has been changed in accordance with the abstract and the conclusions: New Approaches to Determining the D/H Ratio in Aqueous Media Based on Diffuse Laser Light Scattering for Promising Application in Deuterium-Depleted Water Analysis in Antitumor Therapy

Comments 2: The abstract definitely needs to be shortened. Please limit yourself to your own research. Please move information that is important for the manuscript to the introduction section.

Response 2: Thank you, Fixed in Abstract:

«Development of affordable and reliable methods for quantitative determination of stable atomic nuclei in aqueous solutions and adjuvant agents used in tumor chemotherapy is an important task of modern pharmaceutical chemistry. This work quantified the deuterium/prothium isotope ratio in aqueous solutions through an original two-dimensional diffuse laser scattering (2D-DLS) software and hardware system based on chemometric processing of discrete interference patterns (dynamic speckle patterns). For this purpose, 10 mathematical descriptors (di) similar to QSAR descriptors were used. Correlation analysis of bivariate «log di - D/H» plots shows an individual set of multi-descriptors for a given sample with a given D/H ratio (ppm). A diagnostic sign (DS) of differentiation was established: samples were considered homeomorphic if 6 out of 10 descriptors differed by less than 15% (n≥180). The analytical range (r=0.987) between the upper (D/H≤2 ppm) and lower (D/H=180 ppm) limits for the quantification of stable hydro-gen nuclei in water and aqueous solutions was established. Using the Spirotox method, a «safe zone» for protozoan survival was determined between 50 and 130 ppm D/H. Here we discuss the dispersive (DLS, LALLS) and optical properties (refractometric coefficient, optical rotation angle) of the solutions with different D/H ratios that diffuse laser radiation due to surface density in-homogeneities. The obtained findings may pave the way for the future use of a portable in situ diffuse laser light scattering instrument to determine deuterium in water and aqueous adjuvants».

Comments 3: Please limit excessive linking of literature, for example [21-29]. Instead, please provide some examples (line 119-120).

Response 3: Thanks for your valuable comment! The examples described in this paragraph (lines 119-139) are taken from references 21-29.

For a better understanding of this section, the references are organized into topic sentences, according to the example taken about the ddw effect and its literary reference:

Comments 4: To enhance the manuscript, theoretical analysis regarding the stability of the structures for tested water samples in experimental condition is welcome

Response 4: Thank you for this addition!

The stability of structures in aqueous media sensitive to variations in the deuterium content (D/H ratio) and the properties of aqueous solutions with variations in the heavy hydrogen isotope content have been analyzed under experimental conditions in previous articles by the authors:

51. Bunkin, N.F.; Shkirin, A.V.; Babenko, V.A.; Sychev, A. A. ; Lomkova A. K.; Kulikov E. S.; Laser diagnostics of the Bubston phase in the bulk of aqueous salt solutions. Phys. Wave Phen. 2015, 23, 161–175. https://doi.org/10.3103/S1541308X15030012

52.Yurchenko, S. O.; Shkirin, A.V.; Ninham, B. W.; Sychev, A. A.; Babenko, V. A.; Penkov, N.V. Kryuchkov, N.P.; Bunkin, N.F. Ion-Specific and Thermal Effects in the Stabilization of the Gas Nanobubble Phase in Bulk Aqueous Electrolyte So-lutions Langmuir. 2016, 32, 11245-11255.

53. Goncharuk, V.V., Syroeshkin, A.V., Pleteneva, T.V. et al. On the possibility of chiral structure-density submillimeter inhomogeneities existing in water. J. Water Chem. Technol. 39, 319–324 (2017). https://doi.org/10.3103/S1063455X17060029
54. Bunkin, N.; Sabirov, L.; Semenov, D.; Ismailov, F.; Khasanov, M. Nanoscale Structural Phase Transitions in Aqueous So-lutions of Organic Molecules. Condens. Matter 2023, 8, 64.
55. Goncharuk, V.V., Lapshin, V.B., Burdeinaya, T.N. et al. Physicochemical properties and biological activity of the water depleted of heavy isotopes. J. Water Chem. Technol. 33, 8–13 (2011). https://doi.org/10.3103/S1063455X11010024
56. N. F. Bunkin; A. V. Shkirin; V. A. Kozlov; B. W. Ninham; E. V. Uspenskaya; S. V. Gudkov Near-surface structure of Nafion in deuterated water. J. Chem. Phys. 2018,149, 164901 https://doi.org/10.1063/1.5022264

References to the articles and inserted in the text of the manuscript

Comments 5: It is recommended to create a supporting information file containing the original spectra and visualization of the analyses performed in order to validate the conducted research.

Response 5:  Many thanks to the honored reviewer for the fair comment, the file with the supporting data - graphical and tabular - is attached. References to Figures S1,S2,S3 and Tables S1 are given in the text of the manuscript.

Comments 6: Due to the nature of the study, he is considering introducing a future prospective section.

Response 6: We thank the honoured reviewer for his valuable comment, which we have taken into account in the preparation of the final section the prospects of application of the presented development. 

4. Discussion

The importance of developing innovative solutions in the field of physical medicine and rehabilitation is undeniable [Galić, I.; Habijan, M.; Leventić, H.; Romić, K. Machine Learning Empowering Personalized Medicine: A Comprehensive Review of Medical Image Analysis Methods. Electronics 2023, 12, 4411. https://doi.org/10.3390/electronics12214411]. The trend towards the development and implementation of rapid diagnostic systems and compact measurement devices are increasingly affecting the medical and pharmaceutical fields, driven by the recommendations of the WHO and the International Agency for Research on Cancer (IARC https://gco.iarc.fr/today/en) for patient-oriented prevention, early detection, treatment of cancer, adjuvant therapy and palliative care. This article presents the possibilities of a method based on the physical phenomenon of diffuse scattering from the surface of a liquid sample due to the nature and properties of aqueous deuterium-dependent structures of inhomogeneous density. The article explores two promising directions for scaling up the proposed two-dimensional diffuse laser scattering (2D-DLS) method (Figure S3) and expanding the range of deuterium depleted water (DDW) preparations for analysis, as part of a phase II clinical trial to assess the potential usefulness of DDW for therapeutic use (https://hyd.hu/en/home-2/).

Comments 7: Instead of creating an Abbreviation section, please place appropriate abbreviations in the text where they first appear.

Response 7: Thank you for your comments and suggestions. The authors have checked the transcripts of all abbreviations in the text and corrected omissions. We kindly ask the honored reviewer to agree to keep the table of abbreviations at the end of the manuscript.

With gratitude, the authors

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The paper entitled: “Advances in the Development of Methods for Stable Hydrogen Isotopes Determination in Aqueous Solutions for Adjuvant Therapy Based on the Technique of Two-Dimensional Diffuse Laser Light Scattering” by Anton V. Syroeshkin et al. deals with the development of an indirect and speditive analytical technique to determine the concentration of deuterated water in mixtures for various applications.

The paper is full of intriguing and controversial and still unresolved issues both at the basic science level and at the technical (analytical chemistry) level.

On one end the paper is therefore very interesting in our opinion, but on the other hand the number of controversial issues should be ideally reduced and fully addressed in order to be passable for the scientific community.

Moreover, there are many peculiarities in the use of English language and style imperfections which must be addressed  before considering its publication.

On following a list of observations:

Phrases such as : “Due to the relevance of introducing a new concept of therapy into medicine – predictive, preventive and personalized, including, among other things, the use of therapeutics with stable and unstable atomic nuclei,...”

are very generic and should be avoided. Otherwise, each single concept should be fully explained and dealt with. Probably, just by beginning with the rest of the period would be enough for a technical paper, i.e. “... the development of methods for their quantification by accessible and reliable methods is an important task of modern pharmaceutical chemistry. In order to quantify the ratio of deuterium/protium isotopes in aqueous solutions…”

“.. and adjuvant agents used in chemotherapy of tumors,” 

The concept of using hydrogen isotopes in therapy is innovative and controversial and would require full explanation. Possibly in a dedicated review paper. 

Additionally, the concept of “adjuvant therapy” is a very precise one and improperly used in the presented context.

 

“deuterium depletion adjuvant therapy contributes to a better survival rate of patients at the advanced stages of severe oncological pathologies [34]. The anticancer effect of D depletion was confirmed in randomized human phase 2 clinical trial on prostate cancer [35].”

All these controversial clinical concepts should be stated in a less peremptory tone. In fact, citations mostly refer to preclinical studies and deuterium depletion is far from being a consolidated clinical practice.

Despite the fact that we are fascinated by such innovative clinical practice,we think that using stronger than necessary phrasing can be more detrimental than useful in introducing new concepts in medical science and could arouse suspicion more than scientific credibility.

However, since analytical techniques that could determine quantitative D/H ratio can be useful in many applications, the proposed expeditive indirect method is per se interesting.

But the proposed “original chemometric approach to processing dynamic speckle patterns of back diffuse scattering from the surface of liquid objects” is very controversial and would require cross validation by comparison with at least one of the other recognised methods, cited in the Introduction.

Since, as the authors state “the descriptor reflects not only spatial inhomogeneities on the surface or color, but also the dynamic variability of light reflection.” there is an intrinsically strong lack of specificity of the proposed method which should be carefully controlled to provide significant measurements.

Therefore, control experiments able to unveil non-specific components of the signals are strongly needed.

Aside from all so far said, the Spirotox method, described in fancy and particular terms, in fact is a biological test based on the survival rate of a microorganism.

If this test was used as part of a strategy to validate deuterium tests is apparent but not clear in the experimental design. A chart of the experimental design would be useful to insert in the paper.

It is then unclear the definition of “surface scatterers”. Not that scatterers are incomprehensible but why only some of all potential large number have been considered? This is not evident for the average reader scientist, please explain in detail.

Then “particle size distribution” again needs clarification. What particles? Among the many possible sources of particle formation in a complex mixture?

Experimental design was not sufficiently described in methods and consequently graphs are not fully understandable.

Fig. 5, 6,7, 8 are collections of graphs with different axes and scales. Please use homogeneous graphs in Figures and eventually use zooms if you want to show details in each graph

Please do not rush and intertwin concepts in order to allow readers to follow the logical development of experiments.

When dealing with water lattice, particle formation etc.. do not overlap concepts or use encompassing definitions instead.

Fig.10 is “fingerprint” but this “fingerprint” analogy must be carefully explained. FIngerprint is a very specific concept and we seem to get lost in understanding the analogy here.

Conclusions seem not supported by data.

“the properties of these structures were influenced by the isotopic composition of water, namely the ratio of heavy and light hydrogen atoms, which have the greatest difference in masses and thus exhibit a noticeable effect in case of substitution”

but what other possible sources of interference on “dynamic laser speckle” have been evaluated here? Are there possible interferences and / or limitations?

How would the experimental setting be in case of deuterium-depleted water determination in “portable equipment” situations? This remains vague and unclear to us.

 

In conclusion, despite the interest in all these alleged innovations we think the authors failed to describe or rather to demonstrate the validity of their affirmations. However, due to the potential relevance of proposed ideas, we encourage them to better explain and detail their experiments and possibly unpack their ideas in more than one paper.

 

Comments on the Quality of English Language

In need of professional or mother language revision

Author Response

Response to Reviewer 2 Comments

The authors thank the respected Reviewer for valuable comments aimed at eliminating erroneous conclusions, inaccuracies in wording and at improving the quality of the material of the article, in general, to meet the high requirements of Scientia Pharmaceutica.

 Comments 1: Phrases such as :

“Due to the relevance of introducing a new concept of therapy into medicine – predictive, preventive and personalized, including, among other things, the use of therapeutics with stable and unstable atomic nuclei,...” are very generic and should be avoided. Otherwise, each single concept should be fully explained and dealt with. Probably, just by beginning with the rest of the period would be enough for a technical paper, i.e. “... the development of methods for their quantification by accessible and reliable methods is an important task of modern pharmaceutical chemistry. In order to quantify the ratio of deuterium/protium isotopes in aqueous solutions and adjuvant agents used in chemotherapy of tumors,”.

Response 1: The authors agree with the suggestion of the respected reviewer. Fixed in Abstract:

«Development of affordable and reliable methods for quantitative determination of stable atomic nuclei in aqueous solutions and adjuvant agents used in tumor chemotherapy is an important task of modern pharmaceutical chemistry. This work  quantified the deuterium/prothium iso-tope ratio in aqueous solutions  through an original two-dimensional diffuse laser scattering (2D-DLS) software and hardware system based on chemometric processing of discrete interference patterns (dynamic speckle patterns)».

Comments 2: The concept of using hydrogen isotopes in therapy is innovative and controversial and would require full explanation: “adjuvant therapy” is a very precise one and improperly used in the presented context. “deuterium depletion adjuvant therapy contributes to a better survival rate of patients at the advanced stages of severe oncological pathologies [34]. The anticancer effect of D depletion was confirmed in randomized human phase 2 clinical trial on prostate cancer [35].” All these controversial clinical concepts should be stated in a less peremptory tone. In fact, citations mostly refer to preclinical studies and deuterium depletion is far from being a consolidated clinical practice. Despite the fact that we are fascinated by such innovative clinical practice, we think that using stronger than necessary phrasing can be more detrimental than useful in introducing new concepts in medical science and could arouse suspicion more than scientific credibility.

Response 2: The authors thank the respected reviewer for his attention to important details of the manuscript on the effects of D/H ratios in aquatic environments. In reviewing the biological effects of deuterium depleted water, the authors have relied on 35 years of experience in worldwide research on the biological effects of naturally occurring deuterium with human and animal participants, in particular the work of a renowned Hungarian scientist and founder of HYD LLC for Cancer Research and Drug Development https://hyd.hu/en/home-2/ for anti-cancer research and drug development based on a patented deuterium depletion process and initiation of drug registration. The International Congress on Deuterium Depletion https://deuteriumdepletion.com/4th-congress-2019/scientific-program/ with the participation of medical, pharmaceutical, biological, chemical and physical specialists allows to be in the center of research results and achievements on the properties of light water (deuterium depletion from 125 to 25 ppm). Drinking water products with reduced (depleted) deuterium content, licensed in Budapest, are sold in pharmacies in more than 50 countries around the world. And given the preclinical studies conducted by DDW and the absence of significant side effects, these products can be used as a preventive treatment to enhance the main effects of conventional chemotherapy:

1. Somlyai, G.; Kovács, B. Zs.; Somlyai, I.; Papp, A.; Nagy L. I. ; Puskás L. G. Deuterium depletion inhibits lung cancer cell growth and migration in vitro and results in severalfold increase of median survival time of non-small cell lung cancer patients receiving conventional therapy. Journal of Cancer Research & Therapy 2021, 9,12-19.
2. Zhang, X.; Wang, J.; Zubarev, R.A. Slight deuterium enrichment in water acts as an antioxidant: Is deuterium a cell growth regulator? Mol. Cell. Proteom. 2020, 19, 1790–1804.
3. Zhang, X.; Gaetani, M.; Chernobrovkin, A.; Zubarev, R.A. Anticancer Effect of Deuterium Depleted Water—Redox Disbalance Leads to Oxidative Stress. Mol. Cell. Proteom. 2019, 18, 2373–2387
4. Boros, L.G.; D’Agostino, D.P.; Katz, H.E.; Roth, J.P.; Meuillet, E.J.; Somlyai, G. Submolecular regulation of cell transformation by deuterium depleting water exchange reactions in the tricarboxylic acid substrate cycle. Med. Hypothesis 2016, 87, 69–74.
5. Krempels, K.; Somlyai, I.; Gyöngyi, Z.; Ember, I.; Balog, K.; Abonyi, O.; Somlyai, G. A retrospective study of survival in breast cancer patients undergoing deuterium depletion in addition to conventional therapies. J. Cancer Res. Ther. 2013, 194–200.
6. Gyöngyi, Z.; Budán, F.; Szabó, I.; Ember, I.; Kiss, I.; Krempels, K.; Somlyai, I.; Somlyai, G. Deuterium Depleted Water Effects on Survival of Lung Cancer Patients and Expression of Kras, Bcl2, and Myc Genes in Mouse Lung. Nutr. Cancer 2003, 65, 240–246.
7. Somlyai, G.; Laskay, G.; Berkényi, T.; Galbács, Z.; Galbács, G.; Kiss, S.A.; Jákli, G.; Jancsó, G. The biological effects of deuterium-depleted water a possible new tool in cancer therapy. J. Oncol. 1998, 30, 91–94.

 

Because the authors treat the reviewer's opinion and recommendations with the utmost respect, it was decided to present the results on the biological effects of DDW in this manuscript with extreme caution.

Moreover, based on the fair comments of the reviewers, the authors considered it necessary to make a change to the title of the manuscript, which most closely reflects the purpose and objectives of the study: New Approaches to Determining the D/H Ratio in Aqueous Media Based on Diffuse Laser Light Scattering for Promising Application in Deuterium Depleted Water Analysis in Anti-tumor Therapy .

Comments 3: However, since analytical techniques that could determine quantitative D/H ratio can be useful in many applications, the proposed expeditive indirect method is per se interesting.

But the proposed “original chemometric approach to processing dynamic speckle patterns of back diffuse scattering from the surface of liquid objects” is very controversial and would require cross validation by comparison with at least one of the other recognised methods, cited in the Introduction.

Response 3: We’d like to thank the respected reviewer for a fair comment on the need for control experiments and for cross-validation.

The predictive abilities of our proposed chemometric model were evaluated in advance with a set of training data prepared in vitro by dilution of D₂O (99.9%) ddw (≤2ppm) and test data from water samples with different D/H ratios by resubstitution cross-validation. Actual (test) D/H values (ppm) were determined using Off-Axis Integrated Cavity Output Spectroscopy (OA-ICOS) under continuous measurement conditions (accuracy ~0.1% for d¹⁸O, ~1% for d²H); training data were obtained using two-dimensional diffuse laser scattering. The satisfactory statistical accuracy of the forecast gave us the opportunity to testify to the usefulness of the developed approach for determining the D/H ratio in aqueous samples.

Comments 4: Since, as the authors state “the descriptor reflects not only spatial inhomogeneities on the surface or color, but also the dynamic variability of light reflection.” there is an intrinsically strong lack of specificity of the proposed method which should be carefully controlled to provide significant measurements. Therefore, control experiments able to unveil non-specific components of the signals are strongly needed.

Response 4: We kindly thank the respected reviewer for fair questions related to the concepts of scattering objects formed in liquid media. In describing the phenomenon of dynamic speckle formation, we have relied on the phenomenon of temporal evolution of the speckle pattern, which is well known in physics [Rodrigo H. Henao, Hector Jorge Rabal, Alberto A. Tagliaferri, Roberto Daniel Torroba, "Digital display of the temporal evolution of speckle patterns," Opt. Eng. 35(1) (1 January 1996) https://doi.org/10.1117/1.600939; Bingxue Liu, Dmitry Postnov, David A. Boas, and Xiaojun Cheng, "Dynamic light scattering and laser speckle contrast imaging of the brain: theory of the spatial and temporal statistics of speckle pattern evolution," Biomed. Opt. Express 15, 579-593 (2024) https://doi.org/10.1364/BOE.510333; Liu, Y.; Bu, P.; Jiao, M.; Xing, J.; Kou, K.; Lian, T.; Wang, X.; Liu, Y. Dynamic Speckle Illumination Digital Holographic Microscopy by Doubly Scattered System. Photonics 2021, 8, 276. https://doi.org/10.3390/photonics8070276]. Changes of scattering objects in a liquid medium are caused by their movement and subsequent formation of the interference pattern of speckle, where its grains - elements of grayness - change their intensity and shape with the passage of time. However, the time history of the alternation of bright and dark speckles carries visual information about the distribution of scatterers in the liquid medium, since it determines the amplitude and phases of the wave field as a result of the superposition of the reference coherent wave and the waves diffracted by the object. The dynamics of the scatterer distribution can only be related to the nature and properties of the medium, as we have demonstrated using hydrogen isotope variations [Near-surface structure of Nafion in deuterated water /N. F. Bunkin, A. V. Shkirin, V. A. Kozlov, B. W. Ninham, E.V. Uspenskaya, S.V. Gudkov. Journal of Chemical Physics. ‒ 2018 https://doi.org/10.1063/1.5042065; Anton Syroeshkin, Tatiana Pleteneva, Elena Uspenskaya, Igor Zlatskiy, Nadine Antipova, Tatiana Grebennikova, Olga Levitskaya, D/H control of chemical kinetics in water solutions under low deuterium concentrations, Chemical Engineering Journal, Volume 377, 2019, https://doi.org/10.1016/j.cej.2018.08.213;Time dependence of the luminescence from a polymer membrane swollen in water: Concentration and isotopic effects / N.F. Bunkin, G.A. Lyakhov, V.A. Kozlov, A.V. Shkirin, I.I. Molchanov, M.T. Vu, I.S. Bereza, N.G. Bolikov, V.L. Fouilhe, I.S. Golyak, I.S. Golyak, I.L. Fufurin, V.S.Gorelik, E.V. Uspenskaya, H.S. Nguyen, S.V. Gudkov // Physics of Wave Phenomena.─ 2017.─ V. ─ 25. ─№4. ─ Р. 259-271. https://doi.org/10.3103/S1541308X17040045; Goncharuk, V.V., Lapshin, V.B., Burdeinaya, T.N. et al. Physicochemical properties and biological activity of the water depleted of heavy isotopes. J. Water Chem. Technol. 33, 8–13 (2011). https://doi.org/10.3103/S1063455X11010024]

Regarding the specificity of the proposed method, we have previously performed experiments confirming it using a developed mathematical model of folding two-dimensional light scattering (2D-LS) patterns into a descriptor to determine the identification of enantiomers of optically active pharmaceutical substances [Syroeshkin AV, Levitskaya OV, Pleteneva TV, Uspenskaya EV, Tribot-Laspiere MA, Tarabrina IV. The new two-dimensional light scattering method for recognition of pharmaceutical enantiomers. J Appl Pharm Sci, 2020; 10(12):029-034 DOI: 10.7324/JAPS.2020.101204]. Although the problem of optical isomer separation still exists today, the analysis of the composition of optical isomers is not a routine task. 

Comments 5: Aside from all so far said, the Spirotox method, described in fancy and particular terms, in fact is a biological test based on the survival rate of a microorganism. If this test was used as part of a strategy to validate deuterium tests is apparent but not clear in the experimental design. A chart of the experimental design would be useful to insert in the paper.

Response 5: Thank you for your comment and the opportunity to add the design section of the Spirotox Method Research Study:

Spirotox Study Design

Ciliated protozoan Sp. ambiguum (3-5 adults) were placed in a thermostated cell filled with different D/H ratio in water sample. Observation of the behavioral response and fixation of the time of cell death were recorded by successive signs: convulsions - twisting - cessation of motor activity. The temperature (Arrhenius) dependence of the lifetime of Sp. ambiguum was studied (T=301 - 309 K) with subsequent calculation of the observed activation energy of the process of cellular trans-formations described by the schematic diagram (Equation 1).

k = A0 exp,

(1)

Here, A₀ is the pre-exponential factor, Ea is the energy of dead cell stage, R is the universal gas constant, and T is the temperature (K).

The existence of an intermediate state in the process of ligand-induced death of a subject means that the cell death process must occur at the activation stage with energy expenditure (Scheme 1).

Scheme 1. Kinetic scheme of ligand-receptor Sp. ambiguum interaction: C- cell, L-ligand, n-stoichiometric coefficient, C•L_n – intermediate state (cell after interaction with the ligand), K_e is the equilibrium constant fast stage, f_m is the rate constant of the cell transition to the dead state, DC is a dead cell. The inserts show photographs of ciliates at the stages of incubation in the medium and recording of death.

Applying this method of biotesting, we demonstrated the results on finding the range of deuterium content in aqueous medium (50 ppm to 130 ppm), providing not only the duration of survival of test culture, but also high values of activation energy of its death stage - safety zone.

Comments 6: It is then unclear the definition of “surface scatterers”. Not that scatterers are incomprehensible but why only some of all potential large number have been considered? This is not evident for the average reader scientist, please explain in detail. Then “particle size distribution” again needs clarification. What particles? Among the many possible sources of particle formation in a complex mixture?

Response 6: Thank you for your comments and suggestions. According to scientific studies and references, the so-called «surface scattering centres» include Bubston phase, Water clusters, Emulons and others names… causing density fluctuations on different length scales in liquid continuum medium. Terminology is unfortunately not yet unified:

• Gao, Y., Fang, H., Ni, K. et al. Water clusters and density fluctuations in liquid water based on extended hierarchical clustering methods. Sci Rep 12, 8036 (2022). https://doi.org/10.1038/s41598-022-11947-6;
• Bunkin, N.F.; Shkirin, A.V.; Babenko, V.A.; Sychev, A. A. ; Lomkova A. K.; Kulikov E. S.; Laser diagnostics of the Bubston phase in the bulk of aqueous salt solutions. Phys. Wave Phen. 2015, 23, 161–175. https://doi.org/10.3103/S1541308X15030012;
• F. Bunkin; A. V. Shkirin; V. A. Kozlov; B. W. Ninham; E. V. Uspenskaya; S. V. Gudkov Near-surface structure of Nafion in deuterated water. J. Chem. Phys. 2018,149, 164901 https://doi.org/10.1063/1.5022264;
• Goncharuk, V.V., Syroeshkin, A.V., Pleteneva, T.V. et al. On the possibility of chiral structure-density submillimeter inhomogeneities existing in water. J. Water Chem. Technol. 39, 319–324 (2017). https://doi.org/10.3103/S1063455X17060029;
• Zhang, S.; Zhang, Y.; Wu, C.; Yang, H.; Zhang, Q.; Wang, F.; Wang, J.; Gates, I.; Wang, J. A Facile Strategy to Prepare Small Water Clusters via Interacting with Functional Molecules. Int. J. Mol. Sci. 2021, 22, 8250. https://doi.org/10.3390/ijms22158250;
• Yakhno, T.; Drozdov, M.; Yakhno, V. Giant Water Clusters: Where Are They From? Int. J. Mol. Sci. 2019, 20, 1582. https://doi.org/10.3390/ijms20071582].

The dynamics of all described 'scatterers' depend on many properties of water and solutions - pH, dissolved gases, nanoparticles, D/H ratio, etc. The subject of this manuscript is deuterium-dependent density inhomogeneities, which are of the order of (or larger than) the wavelength, are randomly distributed and, as shown experimentally, cause diffuse scattering from the surface.

Comments 7: Experimental design was not sufficiently described in methods and consequently graphs are not fully understandable. Fig. 5, 6,7, 8 are collections of graphs with different axes and scales.

Please use homogeneous graphs in Figures and eventually use zooms if you want to show details in each graph.

Response 7: Thank you! A section on experimental design has been added to the manuscript text:

2.1.2. Study Design

Laboratory test samples with D/H variations from 1 ppm to 200 ppm with a 40 ppm increment were obtained in situ by serially diluting D₂O with DDW aliquots. Water with a D/H ratio of more than 200 ppm was represented by two samples: 1000 ppm and 1·10⁶ ppm (D₂O). For this purpose, micropipettes with volumes from 0.1 µL to 100 µL were used.To exclude extraneous heterogeneous contaminants in the water samples, filtration through a submicron inert membrane filter (Millex-GV Filter with a 0.22 µm pore size hydrophilic PVDF membrane, Merck Millipore, UK).

Scaling of Figures 5-8 brought to uniformity (axis OX).

Comments 8: Fig.10 is “fingerprint” but this “fingerprint” analogy must be carefully explained. FIngerprint is a very specific concept and we seem to get lost in understanding the analogy here.

Response 8: Thank you for your comment! Although the term fingerptint is well known in the Restricted analysis, it is similar in presentation of results and was convenient to use, the authors took note of a respected reviewer’s comment and renamed these graphs into two-dimensional diagrams:

Figure 10. The two-dimensional diagram of the diffuse laser scattering method for water samples with different D/H ratios: water bidistilled (Bd, SMOW-V) and deuterium depleted water (DDW, ≤2 ppm).

Comments 9: Conclusions seem not supported by data. “the properties of these structures were influenced by the isotopic composition of water, namely the ratio of heavy and light hydrogen atoms, which have the greatest difference in masses and thus exhibit a noticeable effect in case of substitution”

but what other possible sources of interference on “dynamic laser speckle” have been evaluated here? Are there possible interferences and / or limitations?

Response 9: Thank you for your comment!

The possible drawbacks of the proposed analytical method, which set limits for application, are as follows: drift of the analyte temperature, reduction of the analyte volume due to the processes of evaporation, exposure to external heterogeneous particles, external vibrations. The interference can cause a shift, increasing or decreasing the signal related to the measured value.

In order to exclude their influence on the suitability of the analytical method for soft (Vidan) work, a functional equivalent to Malvern’s equipment is laid down, according to the preliminary analysis (control of measurement stationarity as a dependence of the descriptor di=f(t,min), presented as Figure 4 in the Manuscript. If the test is successful, the sample shall be measured at a rate of more than 10 frames per second for 60 seconds, with three parallel measurements each with a number of runs (n=180).

Comments 10: How would the experimental setting be in case of deuterium-depleted water determination in “portable equipment” situations? This remains vague and unclear to us.

Response 10: Thank you for your comment! Portable version of equipment using diffuse scattered light technology is presented as an isolated vibration-free working place with emitter emitting high-power laser beam, test sample not subject to additional heating, System lenses, personal computer with installed soft. This equipment is installed on the production and test sites of several pharmaceutical companies in the RF (Figure S3)

 

Figure S3. Portable equipment of diffuse scattered light technology at the production and testing site of the Russian pharmaceutical holding.

Comments 11: Comments on the Quality of English Language In need of professional or mother language revision

Response 11: The manuscrip was proofread by a PhD in linguistics, specialising in academic writing

Author’s team represented by staff - doctors and PhD, the assistants and research assistants of the Department of Pharmaceutical and Toxicological Chemistry of the Medical Institute RUDN are sincerely grateful to the respected reviewer for carefully reading the manuscripts and critical comments. more   

 

With gratitude, the authors

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The Authors have satisfactorily answered all my comments. The manuscript has been improved, reads well, it is well organised and systematic. In my humble opinion, this manuscript should be published in this form in Sci. Pharm.

Author Response

The authors are very grateful to the reviewer for his hard work in reviewing the manuscript, finding errors and mistakes, the correction of which allowed us to significantly improve the quality of the manuscript and hope for its successful publication in Scientia Pharmaceutica.

With gratitude, the authors

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript was improved considerably in its present version compared to the previuos one. Discussion is very short and limitations of the present study should be inserted. Careful review for typos such as "The results" instead of "Results" etc...

Author Response

The authors are very grateful to the honourable reviewer for his hard work in reviewing the manuscript, finding errors and mistakes, the correction of which allowed us to significantly improve its quality and hope for successful publication in Scientia Pharmaceutica.

 

Comments 1: The manuscript was improved considerably in its present version compared to the previuos one. Discussion is very short and limitations of the present study should be inserted.

Response 1: We thank the honourable reviewer for the important comment on the addition of the Conclusion section, which have been implemented as recommended:

«Elimination of the existing drawbacks of the method, such as temperature drift and reduction of analyte volume due to evaporation processes, influence of foreign inhomogeneous particles and external vibrations due to forced temperature control, isolation and other settings of instrumental parameters and measurement modes, will make it possible to eliminate their influence on the suitability of the analytical method and promote its large-scale application [70. Liu, M.; Han, Y.; Xi, X.; Zhu, L.; Yang, S.; Tan, S.; Chen, J.; Li, L.; Yan, B. Multiscale Dense U-Net: A Fast Correction Method for Thermal Drift Artifacts in Laboratory NanoCT Scans of Semi-Conductor Chips. Entropy 2022, 24, 967.] .»

 

Comments 2: Careful review for typos such as "The results" instead of "Results" etc...

Response 2: Thank you, the error has been fixed.

 

With deep gratitude, the authors!

Author Response File: Author Response.docx