Serum Cobalt Concentration and DNA Methylation Signatures in Women with Obesity

Round 1

Reviewer 1 Report

This manuscript reports a pilot case-control study aiming at determining the possible involvement of cobalt, especially serum cobalt concentration, with DNA methylation patterns in women with obesity. For the study, 33 adult women were selected, 17 with normal weight and 16 with obesity. Total leukocyte DNA was obtained from the participants, and processed according to the kit distributor to obtain final DNA methylation values. Serum Cobalt levels were assessed using Inductively Coupled Plasma Spectrometry (ICP-MS). ChAMP was used to to verify the identification of differential methylated positions associated with exposure to cobalt. SPSS software package was used for statistical analysis. The results reported in the manuscript indicate that the functional enrichment analysis of differentially methylated positions (DMPs) highlighted several potential pathways implicated in apoptosis, cancer, metabolic signaling, and obesity, ultimately paving the road for further investigation into the mechanistic links between these pathways and the onset and progression of obesity. 

Comments

The study is properly conducted and the results are properly reported. The conclusion of the authors is supported by the data reported here. 

The insertion of a paragraph describing the limitations of the present study, and how exportable to the general population or other countries are the findings reported in this study would be beneficial, owing to the relative small group of individuals assessed in this pilot study.

Author Response

Thank you for your comment, we have inserted a paragraph describing the limitations of the present study and the extent to which the results reported in this study are exportable to the general population or to other countries. This is in the final paragraph of the discussion and it is highlighted in red.

Reviewer 2 Report

In the present study, the authors investigated the impact of cobalt serum concentration on DNA methylation in women with obesity. For the study, a total of 33 women (17 normoweight and 16 obese) were recruited. Cobalt serum levels were measured by Inductively Coupled Plasma Mass Spectrometry, and the results indicated that normoweight women had higher levels of cobalt in the serum than women with obesity. DNA methylation analysis identified differentially methylated positions (DMPs) associated with Cobalt exposure. Consistent with the observation on Cobalt levels, women with obesity presented with hypomethylation in the DMPs.  Functional analysis suggests that these DMPs are observed in pathways implicated in apoptosis, cancer, and metabolic signaling.

General Comments:  The study appears to be properly formulated and conducted. The conclusions of the authors are supported by the data reported in the manuscript. Overall, the study provides preliminary insights into the interaction between cobalt exposure and DNA methylation in the context of obesity. The mechanisms behind this interaction were not investigated at this time. The limitation of the present study is primarily the small pools of obese women and normo-weight women recruited here. For a preliminary study, the number is sufficient but validation in larger pools is needed. 

 Experimental Design comment: For the study, the authors recruited women with major obesity (BMI = 45.1+/- 5.4), i.e. twice the BMI of the normo-weight. This raises a few questions: 1) why only women with a BMI between 45 and 50 (?) were recruited for the study and not women with lower level of obesity (e.g. BMI more in the 30s Kg/m2)? 2) Based on reported BMI of the obese women, was anything significantly different in the diet or in the abdominal absorption of mineral s that could help explain why the Serum levels of Cobalt were lower in the pathological samples? 3) Were the women with obesity under any type of treatment that could have impacted cobalt absorption?

English: a few typos need to be amended (e.g. abstract, line 28: it would appears that some wording is missing).

An interesting study that aims at determining the role of cobalt in reducing DNA methylation in identified pathways. At the present time the study is  correlative and the reasons for the reduced DMPs in the presence of low serum levels of cobalt were not investigated. A few points need to be further clarified.  

Author Response

 Experimental Design comment: For the study, the authors recruited women with major obesity (BMI = 45.1+/- 5.4), i.e. twice the BMI of the normo-weight.This raises a few questions:

• why only women with a BMI between 45 and 50 (?) were recruited for the study and not women with lower levels of obesity (e.g. BMI more in the 30s Kg/m2)?

Thanks for your comment, this is a very nice question, our selection strategy was driven by the aim to investigate the most pronounced effects of obesity on DNA methylation patterns. Prior research indicates that while DNA methylation changes do occur across various levels of obesity, these changes are often more distinct and potentially more informative in more extreme phenotypes. By selecting individuals at the higher end of the obesity spectrum, we anticipated clearer, more significant differences in methylation patterns, which would be crucial for identifying robust associations. This approach is commonly used in genetic and epigenetic association studies to enhance the detection of relevant biological markers linked to extreme levels of adiposity, thus providing a stronger basis for subsequent analyses and interpretations.

• Based on the reported BMI of the obese women, was anything significantly different in the diet or in the abdominal absorption of mineral s that could help explain why the Serum levels of Cobalt were lower in the pathological samples?

In response to the query about the effects of cobalt on obesity, the study of Kawakami et al. 2012 demonstrates that cobalt chloride (CoCl2) significantly impacts white adipose tissue in mice fed a high-fat diet, reducing adipocyte size and WAT weight. Additionally, cobalt administration notably increased serum leptin, adiponectin, and HDL cholesterol levels, alongside normalizing glucose levels. These findings suggest a beneficial role for cobalt in improving metabolic health markers and activating AMPK in key tissues, which could help prevent obesity-related diseases. This contrasts with the negative effects observed with mercury, underlining cobalt's potential for therapeutic use in obesity management DOI: 10.1016/j.taap.2011.10.004.

The role of cobalt in obesity it is not yet clearly defined, the study of Tetsuka et al. 2022, observed within the Tokyo Teen Cohort a significant inverse correlation between urinary cobalt levels and BMI. These results suggest that cobalt may play a role in reducing obesity risk in early adolescents, particularly males. They state that further research is required to confirm these findings and elucidate the underlying mechanisms (doi: 10.14789/jmj.JMJ21-0043-OA)

We hypotesize that the lower levels of cobalt in individuals with obesity comes from the diet quality of these individuals.
Cobalt is a trace element most commonly known for its vital role in the body as part of vitamin B12, which is crucial for nerve function and the production of DNA and red blood cells. In terms of dietary sources, cobalt is not directly consumed but is intrinsic to the structure of vitamin B12, found in significant amounts in animal products such as meat, fish, dairy, and eggs. While the body only requires small amounts of cobalt, its presence in the diet through vitamin B12 is essential for maintaining healthy metabolic processes, including the synthesis of fatty acids and energy production. The adequate intake of foods rich in vitamin B12 is important for ensuring sufficient levels of cobalt to support these critical bodily functions(10.3390/biology12101335).

We included these points in the discussion section; the new parts are highlighted in red.

3) Were the women with obesity under any type of treatment that could have impacted cobalt absorption?

No the women with obesity were not under any treatment yet, althought this patients were eligible for bariatric surgery based on our University Hospital criteria (based on their BMI).

Reviewer 3 Report

An interesting manuscript focusing on cobalt and obesity in woman

Can the nutritional intake of colbalt be assessed for the participants in this study? This would help to add to the nutritional intake of this study and compare with the serum concentrations and nutritional intake.

Line 48 has two full stops in a row, one should be deleted.

The below paper was not referenced https://link.springer.com/article/10.1007/s11356-021-17861-0

"It is worth noting that the sample was made by convenience", what do the authors mean by this?

Thanks.

Author Response