Environmental Hydrogen Concentration as a Novel Factor Determining Changes in Redox Potential

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript “Environmental hydrogen concentration as a novel factor determining changes in redox potential” by Kiyama is a review article in which the author proposed a new approach for the real-time, non-invasive estimation of the redox potential impacting biological metabolism and reactive oxygen species generation. Here, the authors suggests that hydrogen gas (H2) can be measured in the environmental gas compared to the atmosphere, and may serve as a novel factor for redox potential changes in redox enzymes, impacting biological metabolism and reactive oxygen species generation. In general, this article is critical in this field and contains essential contents. I have minor concerns before this manuscript is accepted for publication.

 

The conclusion was a little bit long. Please summarize it!

 

Abstract: intracellular →　Intracellular

 

Figure 1: SHE should be spelled out.

 

Abstract: The description about 0.00V should be avoided.

Author Response

The manuscript “Environmental hydrogen concentration as a novel factor determining changes in redox potential” by Kiyama is a review article in which the author proposed a new approach for the real-time, non-invasive estimation of the redox potential impacting biological metabolism and reactive oxygen species generation. Here, the authors suggests that hydrogen gas (H2) can be measured in the environmental gas compared to the atmosphere, and may serve as a novel factor for redox potential changes in redox enzymes, impacting biological metabolism and reactive oxygen species generation. In general, this article is critical in this field and contains essential contents. I have minor concerns before this manuscript is accepted for publication.

 The conclusion was a little bit long. Please summarize it!

 Abstract: intracellular →　Intracellular

Figure 1: SHE should be spelled out.

 

Reply:

I appreciate your kind suggestions. I've corrected the sentences according to the comments.

The conclusion is summarized. The spell is corrected. SHE is spelled out.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

In this work the author presents an intriguing approach to get at a complicated problem. Through what I’m sure is excellent use of thermodynamic equations, an approach is described for relating changes in the ratio of H2 molecules found environmentally, i.e. within the body fluids (was my understanding) vs in the atmosphere to ultimately get at a way for monitoring impacts on the redox potential of the body due to the activity of redox enzymes, which may be impacted due to disease, changes in metabolic function, etc.

The authors establish this through several relationships that are described. First the reversible hydrogen electrode potential is influenced (in a logarithmic manner) through both the H2 partial pressure and the H+ activity in the solution. The negative log of H+ activity is the pH of the solution, which can also be thought of as the redox potential when compared against a standard reference solution. In the body, the pH value (and thus H+ activity) is maintained tightly and does not vary. The environmental H2 concentration may however change, and this can be determined through measurements of the end-tidal H2 concentration, which is in equilibrium with the H2 partial pressure of the tissues in the body. This H2 concentration in the breath can then be taken as equivalent to the H2 partial pressure of the tissues.

Rather than H+ activity, this study now proposes to use the negative log of the environmental H2 concentration (as measured through end-tidal measurements) to monitor for changes in the redox potential of the human body. Utilizing the comparison to air for a reference solution.

In general I have no issue with the current work and as I said, find it an intriguing approach. I am not sure how sensitive the bulk redox potential of the human body is to the redox potential changes which are happening extremely locally during protein enzyme catalysis. Likewise, I found the connection between ROS/metabolic function and enzyme function (and therefore potential changes) somewhat unclear. I had a hard time with Section 5 and following it / placing it in the context of the work. Perhaps this section could be revisited and streamlined a bit to highlight the most relevant connections. And finally, could the author add just a sentence or two about future studies/experiments to be done to examine the connections proposed in this work? 

Author Response

In general I have no issue with the current work and as I said, find it an intriguing approach. I am not sure how sensitive the bulk redox potential of the human body is to the redox potential changes which are happening extremely locally during protein enzyme catalysis. Likewise, I found the connection between ROS/metabolic function and enzyme function (and therefore potential changes) somewhat unclear. I had a hard time with Section 5 and following it / placing it in the context of the work. Perhaps this section could be revisited and streamlined a bit to highlight the most relevant connections. And finally, could the author add just a sentence or two about future studies/experiments to be done to examine the connections proposed in this work? 

 

Reply.

I appreciate your kind suggestions. I've corrected the sentences according to the comments.

The connection between ROS/metabolic function and enzyme function (and therefore potential changes) somewhat unclear. So the sentences were rewritten and 2 references were added in section 6 ( 352-360).

“A previous in vivo study demonstrated that 4% H₂ inhalation for 1 hour twice a day for 6 months reduced the body weight of rats and increased the level of metabolites such as G6PD and 6PG (Pentose Phosphate Pathway) and malic acid (TCA cycle and cytosol) involved in reactions that reduced nicotinamide adenine dinucleotide phosphate (NADP⁺) to NADPH. [56] In PFV the oxidative current of glucose 6 phosphate dehydrogenase (G6PD) rapidly increased in the applied potential from +0.2 to +0.8 V vs. Ag/AgCl.[57] Glutathione reductase (GR) catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) using NADPH as the reducing cofactor, and the initial-velocity of GR reached saturation depending on the concentration of NADPH.[58]”

In section 5, the last paragraph (330-332) was rewritten according to the comments.

In section 6, the sentence (368-370) was inserted according to the comments.

“The importance of the enzyme kinetics involved must be addressed in future studies to fully grasp how reversible hydrogen electrode potential influence the cellular redox state and oxidative stress.”

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

In the present review, Kiyama describes the importance of intracellular reduction potential to understanding physiological homeostasis in cells and proposes a new approach for the real-time, non-invasive estimation of the influence of environmental concentration of gaseous H₂ on the reduction potentials of the redox species involved in biological metabolism and reactive oxygen species generation. The manuscript addresses a very interesting topic, since the determination of the reduction potential of biologically relevant redox couples in vivo is crucial for a full understanding of crucial metabolic pathways. Hence the scope and content of the manuscript fully match those of Physiologia. Nevertheless, in my opinion some points need to be further clarified and addressed. Hence, the present version of the review by Kiyama requires major changes to be accepted for publication in Physiologia.

Major points

1. In my opinion, the Nenst equation should be explicitly described at the beginning of Chapter 2. This would make the whole chapter much clearer.
2. Chapter 5, Pages 9-10. It appears to me that the content of this chapter is rather unbalanced, since the effect of O₂ is treated in much greater detail than that of H₂. Therefore, I would suggest adding some more information about the latter. If this is not possible, it should be important to stress that the effect of H₂ is much less understood compared to that of O₂.
3. Page 1, Line 41. Cytochromes are metalloproteins containing one or more heme groups. Therefore, they should not be cited among cofactors.
4. Page 3, Lines 78-80. The author should clearly state that this sentence refers to the reaction cited above.
5. Page 3. Lines 90-92. In my opinion, this sentence is rather unclear. In particular, the relationship between the Purbaix diagram and the reported E values is very obscure.
6. Page 4, Lines 114-115. This sentence is not correct, since pH is defined as -log (activity of H⁺ ions in solution). I strongly suggest correcting it as follows “The pH can be calculated as reported in eq 6, where pH(S) corresponds to the standard pH solution (S) and z is calculated according to eq (7)”.
7. Page 4, Lines 139-143. In my opinion, these sentences are quite unclear and should be rewritten.
8. Page 5, Lines 173-174. In my opinion, this sentence is quite unclear and should be rewritten.
9. Page 5. Line 182. In my opinion, the author should insert a further equation, which clearly describes how ERHE depends on both pH₁ and pH₂.
10. Page 6, Lines 189-190. Cytochromes and cupredoxins are metalloproteins containing heme and copper ions as redox centers. Therefore, the sentence should be corrected replacing cytochromes and cupredoxins with hemes and blue-copper centers, respectively.
11. Page 8, Line 244. It is not clear to me what E°’ does correspond to? If this a new parameter and not a typo, its meaning should be described in the text.
12. Page 8, Line 259. In my opinion, the sentence is unclear.
13. Page 9, Line 279. In my opinion, the sentence should be corrected.
14. Page 10, Lines 515-516. In my opinion, the sentence is unclear.
15. Page 11, Lines 362-363. In my opinion, the sentence should be deleted.

Minor points

1. Page 2, Line 42. I my opinion, substance should be replaced with substrates.
2. Page 6. Lines 213. Redox should be inserted between intrisic and potential.
3. Page 7, Line 214. In my opinion, associated to should replaced with relative to.

Author Response

In the present review, Kiyama describes the importance of intracellular reduction potential to understanding physiological homeostasis in cells and proposes a new approach for the real-time, non-invasive estimation of the influence of environmental concentration of gaseous H₂ on the reduction potentials of the redox species involved in biological metabolism and reactive oxygen species generation. The manuscript addresses a very interesting topic, since the determination of the reduction potential of biologically relevant redox couples in vivo is crucial for a full understanding of crucial metabolic pathways. Hence the scope and content of the manuscript fully match those of Physiologia. Nevertheless, in my opinion some points need to be further clarified and addressed. Hence, the present version of the review by Kiyama requires major changes to be accepted for publication in Physiologia.

 

Reply.

I appreciate your kind suggestions. I've corrected the sentences according to the comments.

Major points

1. In my opinion, the Nenst equation should be explicitly described at the beginning of Chapter 2. This would make the whole chapter much clearer.

The Nernst equation was described in 92-96 and Eq. 3.

 

1. Chapter 5, Pages 9-10. It appears to me that the content of this chapter is rather unbalanced, since the effect of O₂ is treated in much greater detail than that of H₂. Therefore, I would suggest adding some more information about the latter. If this is not possible, it should be important to stress that the effect of H₂ is much less understood compared to that of O₂.

In section 6, the sentence (368-370) was inserted according to the comments.

“The importance of the enzyme kinetics involved must be addressed in future studies to fully grasp how reversible hydrogen electrode potential influence the cellular redox state and oxidative stress.”

 

1. Page 1, Line 41. Cytochromes are metalloproteins containing one or more heme groups. Therefore, they should not be cited among cofactors.

The word of “Cytochromes” was deleted in 43.

 

1. Page 3, Lines 78-80. The author should clearly state that this sentence refers to the reaction cited above.

The redox enzyme was replaced with SDH, which was mentioned above.

 

1. Page 3. Lines 90-92. In my opinion, this sentence is rather unclear. In particular, the relationship between the Purbaix diagram and the reported E values is very obscure.

The Pourbaix diagram was replaced with the Nernst equation (92-96) and E value is determined according to the Nernst equation (97-98). 

 

1. Page 4, Lines 114-115. This sentence is not correct, since pH is defined as -log (activity of H⁺ ions in solution). I strongly suggest correcting it as follows “The pH can be calculated as reported in eq 6, where pH(S) corresponds to the standard pH solution (S) and z is calculated according to eq (7)”.

In A Century of pH Measurement - Pure and Applied Chemistry, (www.iupac.org/web/ins/2004-005-2-500), the concept of pH corresponds only to a notional definition and is unique in the sense that it involves a single ion quantity, the activity of the hydrogen ion (Sørensen 1924), which does not exist on its own and is therefore immeasurable by any thermodynamically valid method. Its evaluation requires extra-thermodynamic conventions. In practice, no single electrode potential can be independently assessed and only differences of potential between two electrodes can be calculated. Sørensen proposed that the pH of an unknown solution (X) is obtained from the potential, E (measured in potentiometric conditions; i.e., null current) of a cell previously developed by Bjerrum (Bjerrum 1906).

In section 2, the sentence (120-124) was inserted according to the comments.

“However, since it is defined in terms of a quantity that cannot be measured by a thermodynamically valid method, eq. 4 can be only a notional definition of pH. [14] The U.S. National Institute of Standards and Technology has defined pH values in terms of the electromotive force existing between certain standard electrodes in specified solutions where the sample (X) and the standard pH solution (S) as follows”

 

1. Page 4, Lines 139-143. In my opinion, these sentences are quite unclear and should be rewritten.

In section 2, the sentences (148-150) were rewritten according to the comments.

“Thus, environmental H₂ partial pressure appears to be a redox potential-specific and sensitive factor of the hydrogen electrode in the solution where pH is buffered. It is hypothesized that the environmental H₂ concentration is a novel factor determining changes in the redox potential of the water solution.”

 

1. Page 5, Lines 173-174. In my opinion, this sentence is quite unclear and should be rewritten.

In section 2, the sentences (181-183) were rewritten according to the comments.

“A negative logarithm function is used to convert very small numbers into more manageable values. The concentration of H⁺ in any solution ranges from 1 mol to 10^-14 mol per liter of solution and pH ranges between 0.00 and 14.00.”

 

1. Page 5. Line 182. In my opinion, the author should insert a further equation, which clearly describes how ERHE depends on both pH₁ and pH₂.

In section 3, the sentence (193-194) and Eq. 13 were inserted according to the comments.

If both the fugacity of H2 and the activity of H+ are not a unit, The potential of RHE is calculated from Eqs. 10 and 11 as  (V) at 25°C.

 

1. Page 6, Lines 189-190. Cytochromes and cupredoxins are metalloproteins containing heme and copper ions as redox centers. Therefore, the sentence should be corrected replacing cytochromes and cupredoxins with hemes and blue-copper centers, respectively.

In section 4, the sentence (200-201) was corrected according to the comments.

“Prominent members of redox centers include cytochromesand cupredoxins with hemes and blue-copper centers, respectively.”

 

1. Page 8, Line 244. It is not clear to me what E°’ does correspond to? If this a new parameter and not a typo, its meaning should be described in the text.

In section 4, the sentences (251-255) were rewritten according to the comments.

“The standard potential of any redox enzyme should be reported with respect to the SHE. The conversion of potential values between a RHE and the SHE can be easily accomplished using the below equation:  where E° is measured versus the SHE; E°’ is the potential measured versus the RHE; and E_RHE is the potential of the RHE versus the SHE (Figure 5).”

 

1. Page 8, Line 259. In my opinion, the sentence is unclear.

In section 4, the sentences (267-273) were rewritten according to the comments.

“When environmental H₂ tension increases and the environmental fluid becomes reduced in spite of pH buffer systems, the electrode potential of the redox enzyme will be considered. An increase in the H₂ partial pressure (environmental concentration) was shown to reduce the redox potential of a phosphate buffer solution according to the Nernst equation.[25]  The electrode potential of the redox enzyme will increase due to an increase in the H₂ partial pressure. Changes in the electrode potential can be calculated wherever pH is buffered as follows:

 

1. Page 9, Line 279. In my opinion, the sentence should be corrected.

In section 5, the sentence (292-294) was rewritten according to the comments.

“During hyperbaric O₂ conditions the magnetic resonance imaging (MRI)-derived extracellular O₂ tension in the brain was markedly lower than the arterial pO_2, but slightly higher than the cerebral venous pO₂.[35]”

 

1. Page 10, Lines 515-516. In my opinion, the sentence is unclear.

In section 5, the sentence (332-333) was rewritten according to the comments.

“In complex I, environmental H₂ influence the direction of electron transport and ROS generation.”

 

1. Page 11, Lines 362-363. In my opinion, the sentence should be deleted.

The sentence was deleted according to the comments.

 

Minor points

1. Page 2, Line 42. I my opinion, substance should be replaced with substrates.

Page 2, Line 42. “Substance” was replaced with substrates.

 

1. Page 6. Lines 213. Redox should be inserted between intrisic and potential.

Page 6. Lines 213. “Redox” was inserted between intrisic and potential.

 

1. Page 7, Line 214. In my opinion, associated to should replaced with relative to.

Page 7, Line 214. “Associated to” was replaced with relative to.

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

In my opionion, the masucript can be accepted for publication in the present form