# Targeted and non-targeted lipidomic analysis of adipose tissue from general population to identify metabolic perturbations related to phthalate exposure

Axel Raux, Celia Pérez-Diaz, Hanne Frederiksen, Francisco Miguel Pérez-Carrascosa, Yann Guitton, Germán Cano-Sancho, Juan Pedro Arrebola

## ► To cite this version:

Axel Raux, Celia Pérez-Diaz, Hanne Frederiksen, Francisco Miguel Pérez-Carrascosa, Yann Guitton, et al. Targeted and non-targeted lipidomic analysis of adipose tissue from general population to identify metabolic perturbations related to phthalate exposure. 16èmes Journées Scientifiques du RFMF, Jun 2024, Saint Malo, France. [hal-04905386](https://hal.science/hal-04905386v1)

HAL Id: hal-04905386

https://hal.science/hal-04905386v1

Submitted on 22 Jan 2025

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Distributed under a Creative Commons Attribution 4.0 International License
# Targeted and non-targeted lipidomic analysis of adipose tissue from general population to identify metabolic perturbations related to phthalate exposure

Axel RAUX<sup>a,b</sup>, Celia PÉREZ-DÍAZ<sup>c,d</sup>, Hanne FREDERIKSEN<sup>e,f</sup>, Francisco Miguel PÉREZ-CARRASCOSA<sup>c</sup>, Yann GUITTON<sup>a,b</sup>, German CANO-SANCHO<sup>a</sup>, Juan-Pedro ARREBOLA<sup>c,d,g</sup>

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LABERCA

Oniris
VetAgroBio Nantes
ÉCOLE NATIONALE

INRAE
la science pour la vie, l'humain, la terre

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MELISA

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a Oniris, INRAe, LABERCA UMR1329, 44037 Nantes, France

b INRAE, MetaboHUB-Grand-Ouest

c Granada University. Department of Preventive Medicine and Public Health, Pharmacy School. Campus de Cartuja s/n, 18071, Granada, Spain

d Instituto de Investigación Biosanitaria (ibs.GRANADA). Avda. de Madrid, 15. Pabellón de Consultas Externas 2, 2ª Planta, 18012, Granada, Spain

e Department of Growth and Reproduction. Copenhagen Univ Hosp, Rigshospitalet, Blegdamsvej 9, 2100, Copenhagen, Denmark.

f International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Copenhagen Univ Hosp, Rigshospitalet, Blegdamsvej 9, 2100, Copenhagen, Denmark

g Consortium for Biomedical Research in Epidemiology and Public Health (CIBERESP), Instituto de Salud Carlos III, C/ Monforte de Lemos 3-5, Pabellón 11. Planta 0, 28029, Madrid, Spain

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## INTRODUCTION

Phthalates are ubiquitous chemical environmental pollutants classified as endocrine disruptors, they are used as plasticizers in different industry applications. In the last decades, some restrictive policies have reduced their use in the industry, but it remains high. They have been associated with obesity, oxidative stress, diabetes or cancer. Adipose tissue dysfunction plays a relevant role in metabolic disorders, however few studies have applied lipidomic analysis to explore the potential associations between environmental chemicals and dysfunctional phenotypes. Neither have these changes been related to different diseases in the long term.

The **objective** of this study was to develop and apply a lipidomic workflow to explore the associations between exposure to phthalates and altered lipid profiles in adipose tissue of general population.

## RESULTS AND DISCUSSION

### Low molecular weight phthalates MnBP and MiBP (ΣMxBP) were associated with a specific lipidomic profile

**Targeted metabolomics:**

15 metabolites were selected based on PLSDA-VIP and all of them were statistically associated with ΣMxBP in adjusted regression models.

**Non-targeted metabolomics:**

216 signals were selected based on PLSDA-VIP and a total of 119 were statistically associated with ΣMxBP, 17 of them were annotated as fatty acids.

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### Non-targeted lipidomics revealed a lipid signature in fat associated with hypercholesterolaemia at 20-years

<figure><img src="image_10.png" /><figcaption>Reduced HDL levels:</figcaption></figure>

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In the non-targeted multivariable logistic analysis, different associations were found between signals and plasma lipid alterations at 20 years of follow-up. Seven of the positive associations found between the signals and the high levels of LDL were also found for the signals and the phthalates.

No previous studies have examined the relationship of phthalates to levels of adipose tissue metabolites and whether these are related to any long-term disease. Therefore, this study is pioneering and its results open the door to further study in this regard.

## EXPERIMENTAL

### GraMo sub-cohort (Spain)
n=81

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<figure><img src="image_13.png" /><figcaption>**Table 1.** Description of the study population divided by the hospital of recruitment.</figcaption></figure>

<table><thead><tr><th></th><th>Granada No. 22</th><th>Motril No. 59</th><th>P-value</th></tr></thead><tbody><tr><td>Age [Median (IQR)]</td><td>49.00 (41.00 - 57.00)</td><td>49.00 (37.00 - 59.50)</td><td>0.94</td></tr><tr><td>BMI [Median (IQR)]</td><td>27.80 (25.30 - 31.96)</td><td>27.80 (24.33 - 30.26)</td><td>0.81</td></tr><tr><td>Sex [n (%)]</td><td></td><td></td><td>0.81</td></tr><tr><td>Women</td><td>11 (50.0)</td><td>32 (54.2)</td><td></td></tr><tr><td>Men</td><td>11 (50.0)</td><td>27 (45.8)</td><td></td></tr><tr><td>Residence area [n (%)]</td><td></td><td></td><td>&lt; 0.0001</td></tr><tr><td>Urban</td><td>5 (22.7)</td><td>3 (5.1)</td><td></td></tr><tr><td>Semi-rural</td><td>1 (4.5)</td><td>45 (76.3)</td><td></td></tr><tr><td>Rural</td><td>16 (72.7)</td><td>11 (18.6)</td><td></td></tr></tbody></table>

### Adipose Tissue Preparation

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### Plasma Preparation

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### Non-targeted Lipidomics

CSH C18 (2.1 x 150 mm; 1.7µm)

Flow Rate : 0.3 mL/min

A : ACN/Water/AmmoAc (60/39/1) + 0.1% AcetAc

B : IPA/ACN/IPA/AmmoAc (88/10/1/1) + 0.1% AcetAc

**Internal Stds**

PC (15:0/15:0)

LysoPC (15:0)

PE (17:0/17:0)

Cer (d18:1/17:0)

TG (17:0/34:0)

C15:0

C23:0

**Q-Exactive**

* Full scan @ 70k with polarity switch

* Iterative DDA Top5 for annotation

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### Phthalates Quantification

Finnigan TSQ Quantum Ultra triple-quadrupole MS

Isotope diluted online-TurboFlow-LC-MS/MS with preceding enzymatic de-conjugation

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### Statistical Analysis

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MWAS-Phthalates
Multiple linear regression
adjusted for age and sex

MWAS Metabolic outcomes
Multiple logistic regression
adjusted for occupation (ldl) and BMI
(hdl)

### Annotations

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Workflow4metabolomics

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GNPS

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hmdb

The Human Metabolome Database

### Pathway Analysis

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## CONCLUSION

In conclusion, our study showed associations between the serum levels of low molecular weight phthalate metabolites (MnBP and MiBP) with a distinctive lipid signature in adipose tissue measured by targeted and non-targeted analysis.

Likewise, some lipids associated with elevated LDL levels and reduced HDL levels at 20 years follow-up. Some lipids were simultaneously associated to phthalate metabolites and prospective hypercholesterolemia at 20 years follow-up, suggesting a potential mediating role. The results of this preliminary study should be considered with caution due to the modest sample size and further confirmatory studies will be required on gain insight on the relationship between phthalates and adipose tissue-related metabolic diseases.

The authors also thank Biogenouest (Western France life science and environment core facility network supported by the Conseil Régional des Pays de la Loire) for supporting MELISA platform (UMR1329), NANTES, France.

The authors from the MELISA platform acknowledge the French National Infrastructure for Metabolomics and Fluxomics MetaboHUB-ANR-11-INBS-0010 (www.metabohub.fr) and the Corsaire metabolomics core facility (Biogenouest). CPR also thanks to INRAE ALIMH for their international mobility grant within the IB20204.