Overview of Capillary Electrophoresis Analysis of Alkaline Phosphatase (ALP) with Emphasis on Post-Translational Modifications (PTMs)

Round 1

Reviewer 1 Report

Introduction: "Detection methods for alkaline phosphatase can be highly sensitive, allowing for the detection of small amounts of the enzyme". Perhaps the authors should mention the reports of single molecule analysis of alkaline phosphatase in 1996 (J. Am. Chem. Soc. 118, 5245-5253) and again in 2000 (J. Am. Chem. Soc. 122, 4853-4855).

Reference 40 was said to "focus on affinity binding for ALP analysis". This seems to be erroneous.

As the title states, the manuscript focuses on the use of capillary electrophoresis for the analysis of alkaline phosphatase. However, there does not seem to be much emphasis on post-translational modifications. 

Author Response

I appreciate your commnets and I highlighted them in the article

Introduction: "Detection methods for alkaline phosphatase can be highly sensitive, allowing for the detection of small amounts of the enzyme". Perhaps the authors should mention the reports of single molecule analysis of alkaline phosphatase in 1996 (J. Am. Chem. Soc. 118, 5245-5253) and again in 2000 (J. Am. Chem. Soc. 122, 4853-4855).

I added them 

Reference 40 was said to "focus on affinity binding for ALP analysis". This seems to be erroneous.

I corrected it

As the title states, the manuscript focuses on the use of capillary electrophoresis for the analysis of alkaline phosphatase. However, there does not seem to be much emphasis on post-translational modifications.

I agree but I tried my best to just give a hint to drive the reader to the future work when capillary electrophoresis techniques developed.

Thanks again.

Author Response File: Author Response.docx

Reviewer 2 Report

This manuscript, review type, focused on Alkaline phosphatase, and the detection using (capillary) electrophoresis. The manuscript is well written, it is easy to read, and to understand. To improve the text, more details of the chemical reactions and methodological diagrams could be included. Additionally, the importance and relevance of alkaline phosphatase in human pathological conditions could be highlighted, and the application of CE en the clinical setup.

Comments:

(1) In the Introduction. The authors could expand the clinical significance of this enzyme.

Fore example, here there are some sentences that may be useful:

"Alkaline phosphatase refers to a group of enzymes that catalyze the hydrolysis of a large number of organic phosphate esters at an alkaline pH optimum. Zinc is an important cofactor. Although alkaline phosphatase is found in many locations throughout the body, its precise function is not yet known.

Alkaline phosphatase activity in serum is derived primarily from three sources: liver (more than 80 percent), bone, and in some patients the intestinal tract.

Serum alkaline phosphatase is derived predominantly from the liver and bones. An elevated alkaline phosphatase can be fractionated to determine if it originates from the liver or bones, although in practice a liver source is usually confirmed by the simultaneous elevation of other measures of cholestasis (eg, gamma-glutamyl transpeptidase).

Other sources may also contribute to serum levels of alkaline phosphatase. Women in the third trimester of pregnancy, for example, have elevated serum alkaline phosphatase levels due to an influx into blood of placental alkaline phosphatase. Individuals with blood types O and B can have elevated serum alkaline phosphatase levels after eating a fatty meal due to an influx of intestinal alkaline phosphatase. Infants and toddlers occasionally display transient marked elevations of alkaline phosphatase in the absence of detectable bone or liver disease. Alkaline phosphatase elevations have been noted in patients with diabetes mellitus. There are also reports of a benign familial occurrence of elevated serum alkaline phosphatase due to intestinal alkaline phosphatase.

The major value of the serum alkaline phosphatase in the diagnosis of liver disorders is in the recognition of cholestatic disease."

(2) In section 2. Regarding the "Background".

It may be useful to show a diagram of the principle of the technique.

This link may be useful:

https://www.genome.gov/genetics-glossary/Electrophoresis

https://www.yourgenome.org/facts/what-is-gel-electrophoresis/

Additionally, a gel with the different isoforms could be shown as well.

For example:

https://www.researchgate.net/figure/Agarose-gel-electrophoresis-of-alkaline-phosphatase-ALP-in-peripheral-blood-PB-serum_fig1_275279128

(3) Regarding capillary electrophoresis. This authors could make a figure to show the basics of the technique.

For example:

https://geneticeducation.co.in/capillary-electrophoresis-of-dna-explained/

(4) Line 162. It would be useful if the authors show the enzymatic reactions of alkaline phosphatase.

For example:

https://www.worthington-biochem.com/products/phosphatase-alkaline/manual

(5) Line 226. Regarding redox reactions. Could you please define this type of reaction (reduction-oxidation)?

(6) Additional information

https://www.clinicallab.com/clinical-applications-of-capillary-electrophoresis-methods-16263

https://doi.org/10.1016/S0009-8981(03)00006-8

Author Response

I appreciate you comments and linkes you provide. they really help me.

This manuscript, review type, focused on Alkaline phosphatase, and the detection using (capillary) electrophoresis. The manuscript is well written, it is easy to read, and to understand. To improve the text, more details of the chemical reactions and methodological diagrams could be included. Additionally, the importance and relevance of alkaline phosphatase in human pathological conditions could be highlighted, and the application of CE en the clinical setup.

Comments:

(1) In the Introduction. The authors could expand the clinical significance of this enzyme.

Fore example, here there are some sentences that may be useful:

"Alkaline phosphatase refers to a group of enzymes that catalyze the hydrolysis of a large number of organic phosphate esters at an alkaline pH optimum. Zinc is an important cofactor. Although alkaline phosphatase is found in many locations throughout the body, its precise function is not yet known.

Alkaline phosphatase activity in serum is derived primarily from three sources: liver (more than 80 percent), bone, and in some patients the intestinal tract.

Serum alkaline phosphatase is derived predominantly from the liver and bones. An elevated alkaline phosphatase can be fractionated to determine if it originates from the liver or bones, although in practice a liver source is usually confirmed by the simultaneous elevation of other measures of cholestasis (eg, gamma-glutamyl transpeptidase).

Other sources may also contribute to serum levels of alkaline phosphatase. Women in the third trimester of pregnancy, for example, have elevated serum alkaline phosphatase levels due to an influx into blood of placental alkaline phosphatase. Individuals with blood types O and B can have elevated serum alkaline phosphatase levels after eating a fatty meal due to an influx of intestinal alkaline phosphatase. Infants and toddlers occasionally display transient marked elevations of alkaline phosphatase in the absence of detectable bone or liver disease. Alkaline phosphatase elevations have been noted in patients with diabetes mellitus. There are also reports of a benign familial occurrence of elevated serum alkaline phosphatase due to intestinal alkaline phosphatase.

The major value of the serum alkaline phosphatase in the diagnosis of liver disorders is in the recognition of cholestatic disease."

I added those information and highlighted them

(2) In section 2. Regarding the "Background".

It may be useful to show a diagram of the principle of the technique.

This link may be useful:

https://www.genome.gov/genetics-glossary/Electrophoresis

https://www.yourgenome.org/facts/what-is-gel-electrophoresis/

Additionally, a gel with the different isoforms could be shown as well.

For example:

https://www.researchgate.net/figure/Agarose-gel-electrophoresis-of-alkaline-phosphatase-ALP-in-peripheral-blood-PB-serum_fig1_275279128

I showed a digram of gel electrophoresis

(3) Regarding capillary electrophoresis. This authors could make a figure to show the basics of the technique.

For example:

https://geneticeducation.co.in/capillary-electrophoresis-of-dna-explained/

I showed a diagram of capillary electrophresis principle and discussed it. 

(4) Line 162. It would be useful if the authors show the enzymatic reactions of alkaline phosphatase.

For example:

https://www.worthington-biochem.com/products/phosphatase-alkaline/manual

that would be very useful information however I did not get enough time to insert it.  

(5) Line 226. Regarding redox reactions. Could you please define this type of reaction (reduction-oxidation)?

I explained and added in the text.

(6) Additional information

https://www.clinicallab.com/clinical-applications-of-capillary-electrophoresis-methods-16263

https://doi.org/10.1016/S0009-8981(03)00006-8

Thanks very much for the information and links you provided, please have the updated version.

Author Response File: Author Response.docx

Reviewer 3 Report

This is really interesting idea to preparaty review on alkaline phosphatase. It can help not only young researchers but also athers to know the full history.

I have no major comments but

1.It will be nice to have ich and wb for alkaline phosphatase to show their cellular localization ( tissue differences?) and molecular mass (species,tissue differences?)

2.In reference to abonentów.It will be nice to have section on antibody history,specyfic charactereistics

Author Response

I appreciate you comments, 

This is really interesting idea to preparaty review on alkaline phosphatase. It can help not only young researchers but also athers to know the full history.

I have no major comments but

1.It will be nice to have ich and wb for alkaline phosphatase to show their cellular localization ( tissue differences?) and molecular mass (species,tissue differences?)

I highlighted the correction in the article.

2.In reference to abonentów.It will be nice to have section on antibody history,specyfic charactereistics.

In this comment, I am planning to have another review paper focusing only on inhibition and there I will consider these headings.

Thanks again

Author Response File: Author Response.docx