Title: Characteristics of serum bile acid profiles among individuals with metabolic dysfunction-
associated steatotic liver disease
Version: 0 Date: 24 Feb 2025
Reviewer:  Akira Honda
Reviewer's report:
This paper addresses the serum bile acid concentrations and compositions in healthy subjects and
MASLD patients. They show that serum total BA concentrations are higher in MASLD patients
than healthy controls. CA, CDCA, and UDCA concentrations are mainly elevated in MASLD
patients. In addition, sex differences in specific bile acid concentrations are also observed. They
conclude that MASLD patients display substantial differences in BA profiles, including sex
disparities. Although valuable data have been obtained on human subjects, data analysis and
discussion that could lead to a better understanding of the pathophysiology of MASLD are
insufficient.
Major comments
1. Since sex differences were observed in Fig. 2B, they focused on sex differences afterward.
However, much more must be compared between MASLD patients and healthy controls, not
males and females. For example, weren't there any differences between patients and controls in
primary to secondary ratio or unconjugated to conjugated ratio?
2. The discussion mentions that CDCA levels in MASLD patients are elevated, as reported in
previous studies. Why would CDCA rise in MASLD? In human BA biosynthesis, the alternative
pathway contributes only 10%. Therefore, CA/CDCA or (CA+DCA)/(CDCA+LCA) ratio is
mainly controlled by CYP8B1. What are these ratios? If they are significantly lower in MASLD
patients, inhibition of CYP8B1 activity is suspected in MASLD.
3. Figure 3A and 3C. What is the relationship between serum total BA concentrations and ALT
levels?
Do male patients with elevated ALT have lower serum total BA concentrations than those with
normal ALT? Could patients with high BA and low ALT have already progressed to MASH?
Minor comments
4. It is now called “metabolic dysfunction-associated steatotic liver disease (MASLD)” instead
of “metabolic dysfunction-associated fatty liver disease (MAFLD).”
5. METHODS/BA Detection section. They mention that detailed information is in the
supplementary material, but I can’t find the supplementary material.
6. Figure 2 and legend. Why do they use both * and #?
7. Figure 3 legend. There is no explanation for the asterisks.
8. Legends of Figure 3A and 3B. They say they compared serum BA profiles between male
and female patients but are comparing normal ALT and elevated ALT patients. "Comparison of
serum BA profiles between normal and high ALT groups in male and female MASLD patients."
may be a better explanation. Figure 3B legend is the same.
9. The last sentence of the second paragraph of DISCUSSION. “BA salt” should be “BA” or
“bile salt.” 7#-dehydroxylation is a multi-step reaction, and a single 7#-dehydroxylase has not
been demonstrated.
10. The second sentence from the end of the fourth paragraph of DISCUSSION. This sentence
is incorrect. Secondary BAs are produced from primary BAs in the gut by microbial multi-step
7#-dehydroxylation.
11. The explanation of the correlation with the "BA profile" is confusing. Correlations with
"some specific BA concentrations" or "individual BA concentrations" would be better. (The last
sentence of the Results section of ABSTRACT, and the fifth paragraph of DISCUSSION)
Title: Characteristics of serum bile acid profiles among individuals with metabolic dysfunction-
associated steatotic liver disease
Version: 0 Date: 24 Feb 2025
Reviewer:  Akira Honda
Reviewer's report:
This paper addresses the serum bile acid concentrations and compositions in healthy subjects and
MASLD patients. They show that serum total BA concentrations are higher in MASLD patients
than healthy controls. CA, CDCA, and UDCA concentrations are mainly elevated in MASLD
patients. In addition, sex differences in specific bile acid concentrations are also observed. They
conclude that MASLD patients display substantial differences in BA profiles, including sex
disparities. Although valuable data have been obtained on human subjects, data analysis and
discussion that could lead to a better understanding of the pathophysiology of MASLD are
insufficient.
Major comments
1. Since sex differences were observed in Fig. 2B, they focused on sex differences afterward.
However, much more must be compared between MASLD patients and healthy controls, not
males and females. For example, weren't there any differences between patients and controls in
primary to secondary ratio or unconjugated to conjugated ratio?
2. The discussion mentions that CDCA levels in MASLD patients are elevated, as reported in
previous studies. Why would CDCA rise in MASLD? In human BA biosynthesis, the alternative
pathway contributes only 10%. Therefore, CA/CDCA or (CA+DCA)/(CDCA+LCA) ratio is
mainly controlled by CYP8B1. What are these ratios? If they are significantly lower in MASLD
patients, inhibition of CYP8B1 activity is suspected in MASLD.
3. Figure 3A and 3C. What is the relationship between serum total BA concentrations and ALT
levels?
Do male patients with elevated ALT have lower serum total BA concentrations than those with
normal ALT? Could patients with high BA and low ALT have already progressed to MASH?
Minor comments
4. It is now called “metabolic dysfunction-associated steatotic liver disease (MASLD)” instead
of “metabolic dysfunction-associated fatty liver disease (MAFLD).”
5. METHODS/BA Detection section. They mention that detailed information is in the
supplementary material, but I can’t find the supplementary material.
6. Figure 2 and legend. Why do they use both * and #?
7. Figure 3 legend. There is no explanation for the asterisks.
8. Legends of Figure 3A and 3B. They say they compared serum BA profiles between male
and female patients but are comparing normal ALT and elevated ALT patients. "Comparison of
serum BA profiles between normal and high ALT groups in male and female MASLD patients."
may be a better explanation. Figure 3B legend is the same.
9. The last sentence of the second paragraph of DISCUSSION. “BA salt” should be “BA” or
“bile salt.” 7#-dehydroxylation is a multi-step reaction, and a single 7#-dehydroxylase has not
been demonstrated.
10. The second sentence from the end of the fourth paragraph of DISCUSSION. This sentence
is incorrect. Secondary BAs are produced from primary BAs in the gut by microbial multi-step
7#-dehydroxylation.
11. The explanation of the correlation with the "BA profile" is confusing. Correlations with
"some specific BA concentrations" or "individual BA concentrations" would be better. (The last
sentence of the Results section of ABSTRACT, and the fifth paragraph of DISCUSSION)
Please review this paper.
user
Title: Characteristics of serum bile acid profiles among individuals with metabolic dysfunction-
associated steatotic liver disease
Version: 0 Date: 24 Feb 2025
Reviewer:  Akira Honda
Reviewer's report:
This paper addresses the serum bile acid concentrations and compositions in healthy subjects and
MASLD patients. They show that serum total BA concentrations are higher in MASLD patients
than healthy controls. CA, CDCA, and UDCA concentrations are mainly elevated in MASLD
patients. In addition, sex differences in specific bile acid concentrations are also observed. They
conclude that MASLD patients display substantial differences in BA profiles, including sex
disparities. Although valuable data have been obtained on human subjects, data analysis and
discussion that could lead to a better understanding of the pathophysiology of MASLD are
insufficient.
Major comments
1. Since sex differences were observed in Fig. 2B, they focused on sex differences afterward.
However, much more must be compared between MASLD patients and healthy controls, not
males and females. For example, weren't there any differences between patients and controls in
primary to secondary ratio or unconjugated to conjugated ratio?
2. The discussion mentions that CDCA levels in MASLD patients are elevated, as reported in
previous studies. Why would CDCA rise in MASLD? In human BA biosynthesis, the alternative
pathway contributes only 10%. Therefore, CA/CDCA or (CA+DCA)/(CDCA+LCA) ratio is
mainly controlled by CYP8B1. What are these ratios? If they are significantly lower in MASLD
patients, inhibition of CYP8B1 activity is suspected in MASLD.
3. Figure 3A and 3C. What is the relationship between serum total BA concentrations and ALT
levels?
Do male patients with elevated ALT have lower serum total BA concentrations than those with
normal ALT? Could patients with high BA and low ALT have already progressed to MASH?
Minor comments
4. It is now called “metabolic dysfunction-associated steatotic liver disease (MASLD)” instead
of “metabolic dysfunction-associated fatty liver disease (MAFLD).”
5. METHODS/BA Detection section. They mention that detailed information is in the
supplementary material, but I can’t find the supplementary material.
6. Figure 2 and legend. Why do they use both * and #?
7. Figure 3 legend. There is no explanation for the asterisks.
8. Legends of Figure 3A and 3B. They say they compared serum BA profiles between male
and female patients but are comparing normal ALT and elevated ALT patients. "Comparison of
serum BA profiles between normal and high ALT groups in male and female MASLD patients."
may be a better explanation. Figure 3B legend is the same.
9. The last sentence of the second paragraph of DISCUSSION. “BA salt” should be “BA” or
“bile salt.” 7#-dehydroxylation is a multi-step reaction, and a single 7#-dehydroxylase has not
been demonstrated.
10. The second sentence from the end of the fourth paragraph of DISCUSSION. This sentence
is incorrect. Secondary BAs are produced from primary BAs in the gut by microbial multi-step
7#-dehydroxylation.
11. The explanation of the correlation with the "BA profile" is confusing. Correlations with
"some specific BA concentrations" or "individual BA concentrations" would be better. (The last
sentence of the Results section of ABSTRACT, and the fifth paragraph of DISCUSSION)
assistant