Lipids are a diverse class of biomolecules with numerous functions, from energy storage to regulating fundamental cellular processes. Oxidized lipids have received much attention currently because they are related to oxidative stress, inflammation, and cell signaling. Oxylipins, for example, are molecules derived from the oxidation of polyunsaturated fatty acids (PUFAs) that are associated with neurodegenerative diseases.

To profile oxylipins in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease, scientists from RIDC Redoxoma led by researcher Sayuri Miyamoto from the Instituto de Química at Universidade de São Paulo (USP), established and validated an ultra-high performance liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) method for simultaneous analysis of 126 oxylipins in plasma. With this tool, they analyzed the plasma of an animal model of ALS and found altered oxylipins in symptomatic animals that reflect oxidative stress, inflammation, and lipid hypermetabolism. According to the researchers, oxylipins can become biomarkers for monitoring disease progression.

“The method was the key to this work. Most methods use low-resolution mass spectrometry to quantify oxylipins and other molecules, a technique with high sensitivity, but not very great characterization power. With the high-resolution mass spectrometry, we have much more accuracy in the characterization of oxylipins and still high sensitivity. We unite the two most relevant aspects and characterize with the maximum possible accuracy a large range of oxylipins”, says Adriano B. Chaves-Filho, who developed the research as a postdoctoral project and is the first author of the article, published in the journal Free Radical Biology and Medicine.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive dysfunction and death of motor neurons in the brain and spinal cord, which leads to muscle atrophy, paralysis, and patient death. Changes in lipid metabolism, chronic inflammation, and oxidative stress are strongly linked to disease progression.

Oxylipins

For the study, the researchers used SOD1-G93A mice, which are transgenic animals that overexpress the human Cu/Zn-superoxide dismutase enzyme gene and develop amyotrophic lateral sclerosis. They examined the plasma of animals at 70 days of age when they are still asymptomatic, and at 120 days when they already show symptoms of the disease. As a control, they used wild-type (WT) animals at the same ages.

“We built a targeted method focused on the quantification of 126 oxylipins. Obviously we didn’t find all of them in the biological sample. We found 56 oxylipins, of which 17 were significantly modified”, says Chaves-Filho.

Figure: Adriano B. Chaves-Filho.

Analysis data revealed that, in the plasma of rats with ALS, oxylipins linked to inflammation and oxidative stress derived from arachidonic acid, such as prostaglandins and monohydroxides, were increased. On the other hand, oxylipins derived from linoleic acid involved in fatty acid absorption and beta-oxidation, called DiHOMES, were decreased.

These DiHOMES are related to the hypermetabolism process, characterized by the mobilization of fatty acids from the adipose tissue for beta-oxidation, due to a greater energy demand, leading to weight loss. In ALS patients, the smaller the amount of adipose tissue, the worse the prognosis. ”Hypermetabolism is a striking feature in people with ALS, who suffer severe weight loss in the final stages of the disease”, says Miyamoto. The researcher’s group will investigate the source of these DiHOMES and their relationship with the development of amyotrophic lateral sclerosis.

Another interesting finding was the increase in ketones derived from arachidonic acid and linoleic acid both in symptomatic animals and the control group at 120 days, showing that age also modulates the metabolism of oxylipins in plasma.

Oxylipins are products derived from the oxidation of polyunsaturated fatty acids, which are targets of oxidation by enzymatic and non-enzymatic mechanisms. According to their chemical properties, oxylipins may have several biological functions, acting as signaling molecules, propagating signals through cells, and modifying biomolecules such as protein and DNA. They may have both pro-inflammatory and pro-resolving activities. One of the most well-established functions of some oxylipins, such as prostaglandins, is inflammatory process mediation.

According to Chaves-Filho, this research “also opens doors in the context of the disease to investigate the mechanisms associated with changes in oxylipins. Because what we did at first was a discovery analysis, which provides information so that new hypotheses can be investigated”.

The researchers point out that performing a global and comprehensive analysis of oxylipins is still challenging. “They are very diverse and have a complex isomerism: many of them share the same molecular formula, the same amount of carbon, oxygen, hydrogen, only the arrangement of these atoms is different”. Added to this is the chemical instability of these molecules, and their low concentration in biological samples.

Hence the importance of the analysis tool, which makes it possible to investigate the profile of oxylipins not only in ALS but also in other pathologies. “It would be interesting if we could do a comparative study of ALS with other neurodegenerative diseases, like Alzheimer’s, for example, and see if the profile is different - because the interesting thing when we do a comprehensive analysis is to establish a profile, a panel of altered oxylipins. We can have different profiles that are very characteristic of each disease”, Miyamoto explains.

The research was carried out with the support of FAPESP and Neurodegenerative Disease Research, Inc. (NDR) and had the collaboration of the groups of researchers Marisa H.G. Medeiros (IQ-USP), Isaías Glezer (UNIFESP), both from RIDC Redoxoma, and William T. Festuccia (ICB-USP).

The article Plasma oxylipin profiling by high resolution mass spectrometry reveal signatures of inflammation and hypermetabolism in amyotrophic lateral sclerosis, de Adriano B. Chaves-Filho, Larissa S. Diniz, Rosangela S. Santos, Rodrigo S. Lima, Hector Oreliana, Isabella F.D. Pinto, Lucas S. Dantas, Alex Inague, Rodrigo L. Faria, Marisa H.G. Medeiros, Isaías Glezer, William T. Festuccia, Marcos Y. Yoshinaga and Sayuri Miyamoto, can be accessed here.