Application of High-Resolution Terahertz Gas Spectroscopy for Studying the Composition of Thermal Decomposition Products in Human Urine of Prostate Cancer Patients

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper shows using a THz spectrometer to identify the composition of urine with prostate cancer as one of the biomarker discoveries. Addressing the comments below would help improve the paper's quality.

1. Please rephrase the paragraph (lines 46-63) describing the previous works. While the authors introduce the previous urine composition study using magnetic resonance spectroscopy (NMR), it is hard for readers to get the points that the previous work shows the importance of whether urine biomarker or the NMR. Also, the description needs to be more organized to read well.

2. Previous work revealed that several compositions were found in urine tests, but it is not clear what the take-home message is.

3. Please add the importance and advantage of using NMR versus conventional state-of-the-art equipment such as GC-MS.

4. The introduction is confusing because this paper reports data from the THz spectrometer, and then there is a more introductory explanation about NMR. Please explain the appropriate connection between THz and NMR and provide additional descriptions of the related background.

5. Please address previous studies identifying the composition in the urine samples with cancers and explain the novelty and differences of this work versus previous urine-based cancer biomarker studies.

6. Data demonstrate the concentrations of several urine compositions compared with reference samples. However, it does not have any analysis perspective. Please explain the key findings from the list of compositions and their concentrations. Analyzing and interpreting the data, in addition to demonstrating, would improve the meaning of the measured data.

7. The number of data shown in the paper is not sufficient. Please consider adding more representative THz spectra. Adding specific schemes or diagrams would help understand how the experiment was done and how the THz spectrometer was used.

Overall, this paper should improve 1) motivation for using the THz spectrometer versus the state-of-art methods, 2) supported data, and 3) critical findings from the conclusion.

Comments on the Quality of English Language

Many sentences have long subjects, which makes them hard to read. The authors need to shorten the subjects.

Many passive voices need to change to active voices.

Generally, many of the paragraphs are wordy and need to be more organized in their structures.

Author Response

Answers  to reviewers

Reviewer 1

1. Please rephrase the paragraph (lines 46-63) describing the previous works. While the authors introduce the previous urine composition study using magnetic resonance spectroscopy (NMR), it is hard for readers to get the points that the previous work shows the importance of whether urine biomarker or the NMR. Also, the description needs to be more organized to read well.

The  paragraph (lines 46-63) describing the previous works is added with some details, The corrections are concerned with description of area where state –of –art prepared, the importance of early diagnostics of  prostate cancer and using the urine in this investigations. These additions are explained further in response to the following comments.

2. Previous work revealed that several compositions were found in urine tests, but it is not clear what the take-home message is.

The note is not sufficiently clear. But we can say that previous work cited in manuscript (Vaks, V.; Anfertev, V.; Chernyaeva, M.; Domracheva,E. et.al. Sensing nitriles with THz spectroscopy of urine vapours from cancers patients subject to chemotherapy. Scientific Reports  2022, 12, 18117 (1-11). https://doi.org/10.1038/s41598-022-22783-z) revealed that several substances were found in urine tests by using THz spectroscopy. The substances (nitriles) appeared in urine in higher concentrations after chemotherapy than before. It can be traces of amino acids from proteins appeared in urine at kidney distortion influenced by platinum-containing chemotherapy.  The last two phrases have been added to manuscript after citing this reference.

3. Please add the importance and advantage of using NMR versus conventional state-of-the-art equipment such as GC-MS.

The advantages and disadvantages of physical methods using in urine investigations have been presented now in manuscript in Introduction.

«The chromatography-mass spectrometry method is as follows: passing through a chromatograph, the sample is separated into components, and the mass spectrometer is responsible for their identification and analysis. Depending on the characteristics of the composition under study and the requirements for the accuracy of the result, one of two methods is used: either high-precision liquid chromatography or gas chromatography with mass spectrometric detection.  The disadvantages of chromatographic methods include the impossibility of simultaneous study of both polar and non-polar substances; this depends on the choice of chromatographic column, as well as the difficulty of sample preparation for a several samples. The main disadvantage of mass spectrometry is that the method is destructive, i.e., it is not the substance itself that is studied, but the products of its transformation. Spectroscopic methods (e.g. nuclear magnetic resonance (NMR), spectroscopies of IR or THz ranges etc.) have more advantages over chromatography-mass spectrometry methods. NMR is a non-destructive method, which makes it possible to carry out repeated experiments with the same sample. NMR requires minimal sample preparation and can detect even previously unknown compounds. Disadvantages of NMR include the difficulty of detecting compounds with high molecular weight (for example, long chains of fats) and the overlap of peaks corresponding to different compounds on each other. Combining NMR with other methods, for example, mass spectroscopy, will improve the efficiency of metabolite identification. Nuclear magnetic resonance will make it possible to find unknown compounds, which can then be targeted in other samples using mass spectroscopy.»

4. The introduction is confusing because this paper reports data from the THz spectrometer, and then there is a more introductory explanation about NMR. Please explain the appropriate connection between THz and NMR and provide additional descriptions of the related background.

The manuscript is devoted to using the THz gas spectroscopy for analysis of urine samples for revealing the differences in molecular content of multicomponent gas mixture of products of thermal decomposition and vapors of urine. First of all it is necessary to reveal what substances were presented in urine at healthy state of human body and pathologies. The Introduction should present State-of –art in area of analysis of urine at diseases including prostate cancer with using various physical methods. Unfortunately, to date, gas spectroscopy methods have practically not been used in urine studies. One of the main methods for studying the composition of urine found in the literature is the NMR method. This fact explains the reason of presentation of  data obtained with using NMR spectroscopy in Introduction. It doesn’t matter in principle by what methods specific  substances were identified, they can be used to create a picture of the composition of urine identified to date. Besides the large molecules determined by NMR allow to propose what small substances can be appeared at thermal decomposition.

The phrase with next explanation has been added to Introduction.

“The analysis of literature data allows to highlight State-of –art in area of analysis of metabolites in urine. Urine should be analyzed first of all for healthy state of human body as well as for states at diseases in particularly at prostate cancer with using various physical methods ”.  

5. Please address previous studies identifying the composition in the urine samples with cancers and explain the novelty and differences of this work versus previous urine-based cancer biomarker studies.

The novelty and differences of this work versus previous urine-based cancer biomarker studies are first of all in using the THz gas nonstationary spectroscopy methods. This method is very sensitive and allows to detect the trace concentrations of substances which can give information about changing the state of human body.  Therefore the composition of product of thermal decomposition of urine at prostate cancer is novel too. 

The manuscript (Conclusions) is added by next phrases:

“The work analyzes the current state of the art in the field of urine examination using physical methods and identifies the methods most often used in such analysis”

The differences in spectral data i.e. presence of absorption lines of some substances (methyl mercaptan, nitriles, formic and acetic acids, pyridine and pyrrole) in data obtained at investigations of urine samples of prostate cancer patients in comparison with healthy volunteers were found for the first time in the world. 

The presented approach is novel for urine analysis and is promising for developing a method for noninvasive study of the composition of biological liquids, which makes it possible to identify metabolite markers of various pathologies and diseases.

6. Data demonstrate the concentrations of several urine compositions compared with reference samples. However, it does not have any analysis perspective. Please explain the key findings from the list of compositions and their concentrations. Analyzing and interpreting the data, in addition to demonstrating, would improve the meaning of the measured data.

The concentrations of substances in this work are not determined. Estimations of ones are discussed qualitatively on the base of presence or absence of more weak absorption lines in measured experimental spectra. This approach to qualitative estimation of concentration is described in last paragraph of Materials and Methods.

The perspective of application the approach presented in manuscript is shown now in last paragraph of Discussion:

The resulting list of substances and the differences in the composition of substances identified for patients with prostate cancer and conditionally healthy volunteers allow, with the accumulation of data, to draw conclusions about significant substances appearing in urine heated to obtain a gas mixture at prostate cancer. In the future, a comparison can be done with the products of thermal decomposition of amino acids, which make up PSA, which increases in urine in prostate cancer. This will make it possible to use the identified markers among the thermal decomposition products of urine to create chemical sensors for specific substances, which can be used to diagnose prostate cancer.

7. The number of data shown in the paper is not sufficient. Please consider adding more representative THz spectra. Adding specific schemes or diagrams would help understand how the experiment was done and how the THz spectrometer was used.

The Figure shown the diagram of experiments was presented in graphical abstract in previous variant and has been added now in manuscript in Materials and Methods.

Overall, this paper should improve 1) motivation for using the THz spectrometer versus the state-of-art methods, 2) supported data, and 3) critical findings from the conclusion.

The corrections carried out in manuscript are improved all items above.  

Comments on the Quality of English Language

Many sentences have long subjects, which makes them hard to read. The authors need to shorten the subjects.

Many passive voices need to change to active voices.

Generally, many of the paragraphs are wordy and need to be more organized in their structures.

The fellow – interpreter who worked on the article has been publishing in English-language magazines for a long time. There are usually no complaints about the quality of the translation. Nevertheless some corrections of text were carried out.

Please see the attachment, where all corrections of manuscript described.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Author,

This is well constructed paper that reports the finding from interesting study. In the main, there are some minor mistakes in the manuscript; You should consider and correct the following points;

·         Page 2 Line 60, ‘’N-methylhistidine’’ Please write the N italic.

·         More detailed information about prostate cancer (such as how many people on average it affects each year) can be provided in the introduction.

·         The conclusion may be examined in more detail. What will be the contribution of this study to the literature? Explain in the conclusion section.

·         What advantages did the performing of high-resolution THz spectroscopy have in this study compared to other methods in the literature?

Comments on the Quality of English Language

These is only a few minor mistakes. 

Author Response

Reviewer 2

• Page 2 Line 60, ‘’N-methylhistidine’’ Please write the N italic.

N and L further in names of some substances are now italic.

• More detailed information about prostate cancer (such as how many people on average it affects each year) can be provided in the introduction.

The next part with information about prostate cancer has been added in introduction.

“According the data from World Cancer Research Fund International  the prostate cancer is the 2nd most common cancer in men worldwide and the 4th most common cancer overall. Consequently, the proportion of prostate cancer diagnosed at a distant stage increased from 3.9% to 8.2% over the past decade. [Rebecca L. Siegel , Kimberly D. Miller , Hannah E. Fuchs ,  Ahmedin Jemal Cancer statistics, 2022// CA CANCER J CLIN 2022;72:7–33 https://doi.org/10.3322/caac.21708] There were more than 1.4 million new cases of prostate cancer in 2020 [https://www.wcrf.org/cancer-trends/prostate-cancer-statistics/]. Zimbabwe had the highest rate of prostate cancer mortality in 2020, followed by Barbados. The Haiti ,Zambia, Jamaica, Trinidad and Tobago, Bahamas, Dominican Republic, Saint Lucia, Côte d’Ivoire are presented in the 10 countries with high prostate cancer mortality also. “

• The conclusion may be examined in more detail. What will be the contribution of this study to the literature? Explain in the conclusion section.

Two phrases, clarifying the details of presented results, were added to conclusions.

The differences in spectral data i.e. presence of absorption lines of some substances (methyl mercaptan, nitriles, formic and acetic acids, pyridine and pyrrole) in data obtained at investigations of urine samples of prostate cancer patients in comparison with healthy volunteers were found for the first time in the world. 

The appearance of nitriles (acetonitrile, butyronitrile, pentannitrile, pentanedienenitrile, benzonitrile, aminopropionitrile) in the urine of prostate cancer patients can be caused by thermal decomposition of amino acids including ones in PCA.

• What advantages did the performing of high-resolution THz spectroscopy have in this study compared to other methods in the literature?

The next part describing the advantage of THz high resolution spectroscopy for analysis data obtained at investigation of biological sample has been added in Materials and Methods.

“The advantage of terahertz gas spectroscopy is high resolution because the absorption lines at working pressure are narrow and overlapped rarely. The line of rotational spectrum is fingerprint of specific substance because such absorption line parameters as central frequency and line strength are determined by molecular structure. It is benefits over the THz Time-domain spectroscopy and IR spectroscopy (where frequency resolution is lower). Besides the detection of only one absorption line is in principle enough for revealing the substance in multicomponent gas mixture, that is benefit over the spectroscopic methods where the spectral lines corresponding the molecular parts or molecular bonds are detected. Some substances of urine sample are volatile and appeared in gas mixture over the sample without heating. The heating of samples in liquid or solid states allows obtaining the gas state of sample. Some of large biological molecules as proteins, sugars, fats are decomposed at heating, but there are specific features in content of resulting gas mixture of products of thermal decomposition characterizing the patient state.”

Please see the attachment, where all corrections of manuscript are described.

Reviewer 3 Report

Comments and Suggestions for Authors

1. The data of ethylene sulfide in sample 3 are missing in Table 2.

2. I think that the vertical line in Table 2 should be removed and made into a traditional three-line table, and the data in the table should be aligned in one direction.

3. Suggest briefly adding the reasons for using thermal decomposition products for analysis.

4.Suggest briefly supplementing the biological connections between the discovered substances and prostate cancer.

5.Suggest using multivariate statistical methods such as PCA and OPLS-DA to analyze the differences between prostate cancer patients and healthy volunteers.

Author Response

Reviewer 3

1. The data of ethylene sulfide in sample 3 are missing in Table 2.

There are no spectral lines of ethylene sulfide in sample 3 therefore there is no ethylene sulfide in this sample in concentrations allowing the detection. The “0” missed in Table 2 has now been added.

2. I think that the vertical line in Table 2 should be removed and made into a traditional three-line table, and the data in the table should be aligned in one direction.

Now the data in the table are aligned in one direction (right side). The Table has now three global  rows (Substances, Prostate cancer patients (sample number), Conditionally healthy volunteers (sample number)). The vertical lines between the numbers of samples have been removed.

3. Suggest briefly adding the reasons for using thermal decomposition products for analysis.

The next part describing the reasons for using thermal decomposition products for analysis as well as the advantage of THz high resolution spectroscopy for analysis data obtained at investigation of biological sample (as above in answer of Reviewer 2) has been added in Materials and Methods.

“The advantage of terahertz gas spectroscopy is high resolution because the absorption lines at working pressure are narrow and overlapped rarely. The line of rotational spectrum is fingerprint of specific substance because such absorption line parameters as central frequency and line strength are determined by molecular structure. It is benefits over the THz Time-domain spectroscopy and IR spectroscopy (where frequency resolution is lower). Besides the detection of only one absorption line is in principle enough for revealing the substance in multicomponent gas mixture, that is benefit over the spectroscopic methods where the spectral lines corresponding the molecular parts or molecular bonds are detected. Some substances of urine sample are volatile and appeared in gas mixture over the sample without heating. The heating of samples in liquid or solid states allows obtaining the gas state of sample. Some of large biological molecules as proteins, sugars, fats are decomposed at heating, but there are specific features in content of resulting gas mixture of products of thermal decomposition characterizing the patient state.”

4. Suggest briefly supplementing the biological connections between the discovered substances and prostate cancer.

The biological connections were discussed in Discussion. The detailed phrase about specific substances has been added in Discussion.

“As it has been noted above the appearance of nitriles (acetonitrile, butyronitrile, pentannitrile, pentanedienenitrile, benzonitrile, aminopropionitrile) in the urine of prostate cancer patients can be caused by thermal decomposition of amino acids including PSA amino acids.”

5. Suggest using multivariate statistical methods such as PCA and OPLS-DA to analyze the differences between prostate cancer patients and healthy volunteers.

The data presented in this article are pilot data demonstrating confirmation of the proposal of a different composition of urine in patients with prostate cancer and in apparently healthy volunteers. In the future, when more data is accumulated, statistical analysis will be carried out.

Please see the attachment, where all corrections of manuscript are described.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors significantly improved the manuscripts based on the comments.

Reviewer 3 Report

Comments and Suggestions for Authors

No comments.