Carolina Silva1,2,3
Tiago Sousa4,5Marisa Pinho4,5Ana Carolina Fontes1,2
Ana Carolina Abrantes1,2
Hugo Brancal3,6,7
Francisco Peixoto8,9
Rosário Domingues4,5
Carlos Viegas1,2,10*- 1Department of Veterinary Sciences, School of Agricultural and Veterinary Sciences (ECAV), University of Trás-os-Montes e Alto Douro (UTAD), Quinta de Prados, Vila Real, Portugal
- 2Animal and Veterinary Research Center (CECAV)—AL4AnimalS, University of Trás-os-Montes e Alto Douro (UTAD), Vila Real, Portugal
- 3Veterinary Hospital of Covilhã, Quinta das Ferreiras, Covilhã, Portugal
- 4Mass Spectrometry Centre, LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal
- 5CESAM, Departament of Chemistry, University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal
- 6Faculty of Health Sciences, University of Beira Interior, Covilhã, Portugal
- 7School of Agriculture, Polytechnic University of Castelo Branco (ESA-IPCB), Quinta da Senhora de Mércules, Castelo Branco, Portugal
- 8Chemistry Center of Vila Real, University of Trás-os-Montes e Alto Douro (UTAD), Quinta de Prados, Vila Real, Portugal
- 9RISE-Health: Health Research Network, Faculty of Medicine, University of Porto, Porto, Portugal
- 10CIVG—Vasco da Gama Research Center, University School Vasco da Gama (EUVG), Campus Universitário, Coimbra, Portugal
Introduction: Periodontal disease is a progressive infectious-inflammatory disease, and, in the case of dogs, it is one of the most frequently diagnosed pathologies. Fatty acids (FA) play a dual role in inflammatory processes, as they have anti-inflammatory and pro-inflammatory functions. The goals of this study were to compare the FA profiles of whole blood in dierent degrees of canine periodontal disease in a single breed, the Portuguese Podengo, and consequently, to analyze the possible variation of these FA with the progression of the disease.
Methods: Whole blood FA values were determined in healthy dogs, dogs with gingivitis and dogs with periodontitis by gas-chromatography-mass spectrometry. The study sample included 10 dogs considered clinically healthy, 10 dogs with gingivitis and 9 dogs with periodontitis.
Results: Arachidonic acid and omega-3 docosapentaenoic acid were significantly higher in periodontitis cases compared to the control group, but curiously the arachidic acid was lower in the gingivitis group compared to the control group. Total saturated FA was significantly lower in the periodontitis group compared to the control group, while the total polyunsaturated FA was significantly higher in gingivitis and periodontitis groups than in the control group. Omega-6 polyunsaturated FA was significantly higher in cases of periodontitis than in healthy dogs.
Discussion: The results presented suggest that the systemic impact of canine periodontal disease is partly reflected in the lipidomic profile of whole blood FA. The potential roles of the FA identified have important implications for a better understanding of the pathogenesis of canine periodontal disease, being equally endowed with potential as biomarkers for diagnosis and disease progression.
1 Introduction
Periodontal disease (PD) is a progressive infectious-inflammatory disease resulting of an interaction between the host, the microbial biofilm of dental plaque and environmental factors (1, 2). Its staging is based on clinical observation of gingivitis and periodontitis, and can be classified based on the severity of clinical signs and lesions in four stages: PD 1 (gingivitis), PD 2 (early periodontitis with less than 25 % of attachment loss or at most), PD 3 (moderate periodontitis corresponding to 25–50 % of attachment loss), and PD 4 (advanced periodontitis characterized by more than 50% of attachment loss), according the American Veterinary Dental College (3). Gingivitis, the first and reversible phase of the disease, corresponds to inflammation confined to the gingival tissue. As the clinical picture worsens, gingival redness and swelling may cause ulceration and spontaneous bleeding (4). Periodontitis, on the other hand, is a chronic and irreversible inflammation of the periodontal tissues, namely the gums, cementum, periodontal ligament, and alveolar bone. This stage can lead to tooth mobility, tooth loss, bone resorption and osteomyelitis of the maxilla and/or mandible (4–6). There is a progressive nature between the two phases when it is not diagnosed and/or treated in a timely and effective manner (4–21).
The systemic impact of PD is real and widely recognized in human and veterinary medicine (6, 22). Given the significant systemic impact of this disease, Tamura et al. (23) developed a method for estimating periodontal pocket surface area, which has shown potential for assessing the effects of periodontitis on systemic conditions. Research into indices such as this or potential biomarkers is crucial for determining more accurately the relationship between canine PD and systemic inflammation. It is believed that this disease affects almost half of the world's human population (24, 25). In the case of dogs, canine PD is also one of the most prevalent pathologies in small animal clinics (4, 7, 26, 27), which is why it is of great interest. (1, 6).
Lipids are an essential class of molecules that are present in organisms. Its main biological functions are the constitution of cell membranes, and energy storage. Furthermore, they are essential as signaling molecules (28).
In the specific case of fatty acids (FA), their influence on cellular inflammatory responses is recognized), with the inflammatory process resulting from their incorporation into cell membrane phospholipids (29–33).
Furthermore, FA are considered important nutritional factors, and they have been implicated in the pathogenesis of periodontal disease (PD) (34). Different FA have been correlated to pro-inflammatory and anti-inflammatory properties, appearing to have a dual role in inflammation (33), with special emphasis on omega-3 (n-3) and omega-6 (n-6) FA (12, 17, 35–66).
In the case of PD, the interest of polyunsaturated FA (PUFA) is notable in studies carried out in both human (17, 25, 34, 55, 57, 67) and veterinary medicine (12). Specifically, n-3 PUFA have been associated with antibacterial, anti-inflammatory and inflammatory bone loss regulating effects, which justify their research interest in regulating periodontitis (25, 68–70), in addition to their beneficial and preponderant clinical role in pathologies with a marked inflammatory component (12), not only in the initial phase but also during resolution (12, 71, 72). Retecka et al. (23, 73) developed a study in which treatment with n-3 PUFA in PD promoted significantly lower rates of bleeding on probing, as well as other advantages (25, 74). Furthermore, EPA and DHA inhibit potential periodontal pathogens, such as Porphyromonas gingivalis (12, 68, 75). On the other hand, in the specific case of canine PD, a study by Lourenço et al. (12) showed that EPA and DHA supplementation had no benefits in terms of gingival score or disease progression. Therefore, these different results promote an additional need for research in this area. As far as the AA is concerned, this FA is involved in the process that results in bone loss in experimental periodontitis (76, 77). Data like this demonstrates the potential of FA as biomarkers of canine PD, not only in terms of diagnosis, but also in terms of disease progression and treatment. The FA can be analyzed through their profile, which is an integral part of the analysis of the individual's lipidome, which is increasingly the subject of research interest in veterinary medicine (32, 33, 73, 76–78), and will certainly be one of the components of personalized veterinary medicine in the future.
To the best of our knowledge, no study has been published about the profiles of FA in different stages of canine PD, especially in a single breed. Therefore, the aims of this study were to compare the FA profiles in whole blood, determined by gas-chromatography-mass spectrometry (GC-MS), in healthy dogs, dogs with gingivitis and dogs with periodontitis and, consequently, to analyze how the progression of the disease may affect the FA present, all of this based on their potential as biomarkers. This will, therefore, be the first approach to lipidomics in canine PD.
Among other possibilities, whole blood is one of the samples in which the FA profile can be investigated to determine the structural and functional molecular components and their respective quantity. The reason we chose to analyze the whole blood in this study is to simplify the analysis of this sample, especially since this is a first approach to this disease. Thus, there was no need to go through the centrifugation stage, after which we would still have to collect the plasma (79). In addition, this type of sample allows for the analysis of lipid variations at the cellular level (79).
2 Material and methods
2.1 Data collection
This study was approved by the Ethics Committee of University of Trás-os-Montes e Alto Douro (Ref. Doc74-CE-UTAD-2024) and was developed in cooperation with Veterinary Hospital of Covilhã, University of Aveiro and University of Trás-os-Montes e Alto Douro. The study sample comprised 29 medium-sized Portuguese Podengo dogs, which were included in three different groups, depending on the degree or absence of PD, i.e., healthy, with gingivitis or with periodontitis. The owner of each of the animals included in the sample was asked to sign an informed consent form authorizing the use of their animal's data in this study. Furthermore, given the characteristics of this investigation, the ARRIVE guidelines (80) were considered and followed.
For clinical reasons unrelated to this investigation, the blood samples were collected by puncturing the jugular veins and placed in tubes containing heparin anticoagulants and ethylenediamine tetra-acetic acid (EDTA). After that, they were processed promptly. Every animal was required to fast for 12 h. In the first instance, a complete blood count and a serum biochemical profile were carried out on each of the animals in order to detect any analytical alterations that would make it impossible to include them in this study. Therefore, only animals with no analytical alterations, as well as no detectable alterations on clinical examination, were considered for the sample in question. These analyzes were performed using automatic analyzers (BC-5000 Vet, Mindray Fujifilm Portugal, S.A., Vila Nova de Gaia, Portugal and Dri-Chem NX500V, Fujifilm Portugal, S.A., Vila Nova de Gaia, Portugal). The excess sample was frozen at −80 °C for 2 months, after which it was properly processed for FA analysis. Whole blood FA analysis was performed at the Chemistry Research Center of the University of Trás-os-Montes and Alto Douro and Mass Spectrometry Center at the Chemistry Department of the University of Aveiro by GC-MS.
2.2 Inclusion criteria
Based on their PD condition—healthy, gingivitis, or periodontitis—the animals were divided up into three groups. All the individuals had to be vaccinated, dewormed, and have a normal general clinical examination. They also had to be free of any evident analytical abnormalities that would indicate an endocrine, hepatic, infectious, neoplastic or renal disease.
2.3 Exclusion criteria
Exclusion criteria included being under 1 year old, having a systemic disease, receiving medical treatment, and being a female in heat, pregnant, or lactating. In addition, we also considered the fact that no animal would be allowed to participate in this study if it had undergone periodontal therapy or surgery within the preceding 12 months. In addition, no supplementation of n-3 FA could have been given to these dogs during the same previous period of time.
2.4 The characterization of each of the three groups under study
Presence of gingivitis, plaque, and any obvious signs of gingival recession, furcation or root exposure, and missing or moving teeth were visually evaluated in conscious dogs during a clinical examination. After this assessment, each animal was assigned to one of the three groups under study.
The first group, known as the control group, included 10 dogs. None of them showed any signs of dental calculus or plaque. Gingival inflammation was not observed, and the height and architecture of the alveolar margin remained unchanged.
The gingivitis group consisted of 10 dogs, which showed clinical signs compatible with this stage of canine PD. The members of this group had superficial gingival inflammation associated with oedema and marginal erythema. They could additionally exhibit slight spontaneous bleeding or bleeding on touch. No loss of insertion was acceptable, and the alveolar margin's size and architecture had to be normal. It should be noted that a slight accumulation of dental calculus can be seen.
The last group in this study corresponds to dogs diagnosed with periodontitis, thus comprising the periodontitis group, with a total of 9 dogs. In this case, the animals in question had to show classic signs of periodontitis, namely gingival recession, furcation exposure, tooth mobility or even tooth loss, in addition to those already mentioned for the gingivitis group.
2.5 . Lipid extraction from the samples
The lipid extraction was performed using a double extraction procedure using a solvent combination of methanol, chloroform, and water in final proportions of 2:1/0.8 (V/V/V), based on methods previous described by Bligh et al. (81) with some modifications (82). In summary, 200 μL of the samples were transferred to a glass tube to which 750 μL of cold methanol (MeOH) was added. Each tube was vortexed for 20 s and then sonicated for 20 s on ice. Following this, 2.5 mL of cold chloroform was added, and the tubes were incubated for 30 min on ice under shaking (75 rpm) with a magnet every 15–30 min. After 30 min incubation, 625 μL of Milli-Q water was added to each tube, vortexed for 20 s, and a further 10 min incubation on ice at 75 rpm. Finally, the extracts were centrifuged at 2,000 rpm for 5 min, allowing the lipid-rich lower phase to be collected in a new glass tube. The extracts were then dried under a stream of nitrogen, and the lipid residue was stored at −20 °C until quantification.
2.6 . Fatty acid profile from whole blood lipidome analysis
Whole blood in EDTA as anticoagulant was used to analyze the FA content by GC-MS following transmethylation. Thirty microgram aliquots of the lipid extracts were transferred to glass tubes and dried under a nitrogen stream. The lipid films were then dissolved in 1 mL of n-hexane containing C19:0 as an internal standard (1 μg mL−1, CAS number 1731-94-8, Merck, Darmstadt, Germany).
Each tube was added 200 μL of a potassium hydroxide (KOH) 2 M solution in methanol and the mixture was vortexed for 2 min. Next, 2 mL of a saturated sodium chloride (NaCl) solution was added, and the mixture was centrifuged for 5 min at 626 × g to promote phase separation. Cholesterol in the upper (organic) phase was removed using a 10 mm silica column in a pipette tip with wool pre-conditioned with 5 mL of hexane. Methyl esters were added to the column and eluted with a hexane and diethyl ether (95:5, v/v, 3 mL) mixture, then completely dried under a nitrogen stream. Finally, fatty acid methyl esters (FAME) were dissolved in 100 μL of n-hexane, and 2 μL of the resulting solution was injected into an Agilent Technologies 8860 GC System (Santa Clara, CA, USA) equipped with a DB-FFAP column (30 m length, 0.32 mm internal diameter, and 0.25 μm film thickness, J&W Scientific, Folsom, CA, USA).
The gas chromatograph was connected to an Agilent 5977B Network Mass Selective Detector, operating with electron impact ionization at 70 eV, scanning the mass range of m/z 50–550 in a 1 s cycle in full scan mode. The oven temperature program started at 58 °C for 2 min and increased by 25 °C min−1 to 160 °C, by 2 °C min−1 to 210 °C, and by 30 °C min−1 to 250 °C, holding for 10 min. The injector and detector temperatures were set at 220 and 280 °C, respectively. Helium was used as the carrier gas at a flow rate of 1.4 mL min−1. FA were identified by comparing retention times to those of commercial FAME standards in the Supelco 37 Component FAME Mix (ref. 47885-U, Sigma-Aldrich, Darmstadt, Germany) and by MS-spectrum comparison with chemical databases (Wiley 275 library and AOCS lipid library).
The relative percentages of FA were calculated using the percent relative area method.
2.7 . Statistical analysis
Statistical analyzes were performed using Jamovi statistical software (version 2.3.28). The continuous variables were assessed for normality using the Shapiro-Wilk Test. The values of each variable under study were analyzed using one-way analysis of variance (ANOVA, for data with a normal distribution) or the Kruskal–Wallis test (for data with a non-normal distribution). Values compared using one-way ANOVA were subjected to the Tukey or Games-Howell (FA 15:0, FA 16:0, FA 18:1n-7, FA 18:2n-6, FA 20:1n-9, FA 20:3n-6, FA 20:4n-6, FA 20:5n-3, FA 22:5n-6, total PUFA and n-6 PUFA) post hoc test; values compared using the Kruskal-Wallis test were subjected to the Dwass-Steel-Critchlow-Fligner (FA 14:0, FA 16:1n-7, FA 16:1n-9, FA 18:0, FA 18:1n-9, FA 18:3n-3, FA 20:0, FA 20:2n-6, FA 22:5n-3, FA 22:6n-3, total of saturated fatty acids (SFA), total monounsaturated fatty acids (MUFA) and n-3 PUFA) post hoc test. Differences between the study groups in terms of gender, age and weight were analyzed using Fisher's exact test, the Kruskal Wallis test and the one-way ANOVA, respectively. A p-value < 0.05 was considered statistically significant
3 Results
3.1 Study population
Three groups were defined for analysis in this study. The control group (C group) included 10 dogs, none of which showed any signs of plaque or gingival inflammation. In addition, the height and architecture of the alveolar margin remained unchanged. In turn, the 10 dogs belonging to the gingivitis group (G group), as the name suggests, showed clinical signs compatible with this stage, i.e., superficial gingival inflammation associated with oedema and marginal erythema, with the possible presence of dental calculus. In these cases, there was no loss of attachment, and the size and architecture of the alveolar margin were normal. The 9 dogs in the periodontitis group (P group) showed signs of this stage such as gingival recession, furcation exposure, tooth mobility or even tooth loss.
The main characteristics of the study sample (n = 29) are shown in Table 1. It should be noted that none of the dogs included in the sample were neutered. With regard to gender, it was possible to see that there was no statistically significant association between this variable and the stage of canine PD (p = 0.893), i.e., based on the classification of each of the three groups in the sample analyzed. Statistical analysis showed that there were also no statistically significant differences between the groups in terms of age (p = 0.394) and weight (p = 0.089).
Table 1. General characterization of study sample (n = 29).
3.2 Fatty acid-profiling from whole blood
A total of 19 FA were identified and quantified in the whole blood of 29 dogs, belonging to three different groups, according to their degree of PD. The FA identified (Table 2) were myristic acid, pentadecanoic acid, palmitic acid, 7-hexadecenoicacid, palmitoleic acid, stearic acid, oleic acid, vaccenic acid, linoleic acid (LA), α-linolenic acid, arachidic acid, gondoic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, AA, EPA, n-6 DPA, n-3 DPA and DHA. The relative quantification of FA obtained in the three different groups, as well as the respective statistical analyzes, are described in Table 2, where different letters represent statistically significant differences. The graphical representation of these same results can be found in the Supplementary material.
Table 2. Fatty acids profiling identifies by GC-MS after alkaline trimethylation and expressed as relative abundance % obtained in control, gingivitis and periodontitis groups.
The most abundant FA was stearic acid (FA 18:0) followed by palmitic acid (FA 16:0). At the opposite end, the lowest abundant FA detected was α-linolenic acid (FA 18-3). To date, and to the best of the authors' knowledge, no study has been carried out with similar characteristics to this one, particularly in terms of the type of sample and with the same profile of FA as this one. The studies carried out so far on the same species are based on erythrocyte membrane lipidome (33, 77), in which a cluster of 10 FA were identified, so an objective comparison with the results presented here is partly unfeasible. Even so, as far as the control group is concerned, the individual values of most of the FA are in the same order of magnitude as those mentioned in the other articles also developed in the canine species but in the erythrocyte membrane (33, 77).
The statistical analysis revealed that are changes in the profile of some FA, namely arachidic acid, AA, EPA and n-3 DPA. Arachidic acid showed a significantly lower abundance in gingivitis group when compared to the control group. In contrast, AA was significantly higher in periodontitis group than in control group. Regarding of EPA, although a statistically significant value was obtained in the ANOVA test, after carrying out the Games Howell test it was possible to verify that there were no statistically significant differences between the three groups. In relation to the n-3 DPA, the abundance obtained in the periodontitis group was significantly higher than in the control group. The individual FA not mentioned above showed no statistically significant differences between the three groups.
The total FA contents of the FA groups, namely total SFA, total MUFA, total PUFA, n-3 PUFA and n-6 PUFA were calculated for the three different groups, as well as the statistical analysis, are summarized in Table 3, which shows some statistically significant differences. Total SFA was significantly lower in periodontitis group than in control group. Regarding the total PUFA, the abundance obtained in gingivitis and periodontitis groups is significantly higher than in control group. N-6 PUFA was significantly higher in the periodontitis group than in control group. The other total FA contents showed no statistically significant differences between the groups. As with the individual analysis of the FA profile, these results are also represented graphically in the Supplementary material.
Table 3. Total fatty acid contents in control, gingivitis and periodontitis groups.
4 Discussion
Previous research in PD evidenced on the interplay of FA and inflammatory responses in periodontitis (25, 34, 67, 79, 83–92). The first FA for which there were statistically significant differences between the groups was arachidic acid, which showed a significantly lower abundance in gingivitis group when compared to that obtained in control group (90, 93–96). This preliminary result deserves further research in order to understand in more detail the role of this FA in canine PD. Furthermore, given the origin of arachidic acid from dietary sources, it may be important to analyze the individual's diet in detail in future investigations, since the reason for these differences may not only be due to the PD but also to the dietary sources. However, given the breed selected for this study and its aptitude for hunting, it is impossible to collect concrete and objective dietary data, since we know that there may always be uncontrolled food intake on the part of the owners. Besides all this, for a first approach to the profile of FA in canine PD, analyzing the diet was not one of the main objectives of this study.
AA was significantly higher in periodontitis group than in control group. This significantly higher abundance in cases of canine periodontitis was similar to that obtained in different studies carried out in humans (97–99). The combination of these results from several studies implies a probable involvement of high levels of AA in the pathogenesis of periodontitis (97).
AA plays a preponderant role in the inflammatory process, as it is involved in modulating its intensity and duration (34, 55, 97, 100–103), and its metabolism is directly affected by inflammation and oxidative stress (34), both so characteristic of canine PD. In our case, the obtained result suggests the central role that AA plays in the course of an inflammatory process, as PD, and how notorious its elevation is in these cases (34, 55, 97, 104, 105). These facts suggest that AA may have potential as a diagnostic biomarker for canine PD, although more studies are needed to confirm this possibility.
However, it would be a somewhat reductive view to consider that the abundance of AA in periodontitis group, compared to control group, is due exclusively to the consequent release of its best-known inflammatory mediators. This is because AA also plays a role in regulating inflammation, independently of its metabolites, demonstrating protective effects in inflammatory diseases mediated by the immune system (86). Moreover, AA has the ability to destroy bacteria by compromising their cell membranes (86, 106, 107), and as is well known there is a component of bacterial proliferation in oral cavity of dogs with canine PD (108, 109). For all these reasons, the authors of this study also hypothesized that the results relating to AA could also be due to its regulatory capacity in inflammation and its protective effect in immune-mediated inflammatory pathologies, such as canine PD (33, 86, 110–115).
The last FA in which there were statistically significant differences between the groups was the n-3 DPA, with the n-3 DPA in periodontitis group being significantly higher than in control group (32, 116). N-3 DPA has a role in the resolution of inflammation because, like EPA and DHA, it is a substrate for the synthesis of specific pro-resolving mediators with biological activity, such as resolvins and protectins (32, 117, 118). Our result is in line with that described in the study conducted by Figueredo et al. (97), which also obtained significantly higher n-3 DPA abundance in patients with generalized chronic periodontitis. However, in this author's case, the comparative term were patients with gingivitis only (25, 32, 34, 97, 118).
Considering the groups of SFA, MUFA and PUFA, some differences were observed.
The total SFA were significantly lower in periodontitis group than in control group. The SFA induce inflammatory signaling by stimulating TLR 2 and 4 (33, 119). TLR recognize pathogen-associated molecular patterns (PAMP) and trigger innate immune responses to protect the host from invasive pathogens (119). However, one of the peculiarities of some TLR is that they can be activated by endogenous non-microbial molecules, such as SFA, which consequently induces inflammation (119). In the case of canine PD, periodontitis is the degree to which a strong inflammatory component characterizes it. Therefore, it is not to be expected that in the control group, this high total SFA abundance would be synonymous with a potential activation of TLR and, consequently, translate into the induction of inflammation. However, the mechanism by which SFA induce TLR 2 and 4 is not so clear and explicit (119). It is therefore important to explore this, together with the present result, in future studies (25, 33, 55, 120).
The total PUFA in the gingivitis group and the periodontitis group is significantly higher when compared to the control group. In addition, it is possible to denote a significantly higher abundance of n-6 PUFA in individuals with periodontitis, which consequently contributed to the total PUFA being significantly higher especially in individuals with periodontitis, in addition to cases of gingivitis, compared to those clinically healthy (34).
Therefore, the known pro-inflammatory effect of n-6 PUFA should be emphasized (25, 121–123). Sztolztener et al. (124) suggested that changes in AA levels could be useful as indicators of the irreversible progression of inflammation. In our case, the AA value was also significantly higher in periodontitis group compared to control group. Furthermore, in the specific case of PGE2 and LTB4, eicosanoids originating from the metabolism of AA and promoters of inflammation (25, 125), these lipid mediators are associated with bone resorption, collagen destruction, oedema, inflammatory cell recruitment and pain, all of which are characteristics of PD (12, 126). These facts allow us to justify the results obtained in this study, both in terms of AA and n-6 PUFA, indicating the chronic inflammatory nature of this phase of canine PD (4, 83, 127).
It is important to emphasize that if we investigate a breed other than the Portuguese Podengo, in this case, the results presented may be different. This fact has already been shown in Doberman Pinschers and Boxers, due to obtaining different plasma concentrations of n-3 FA (73). It is, therefore, essential that, in studies of this kind, the sample in question is limited to a single breed, and a sample of different breeds should be avoided.
5 Limitations
Firstly, the main limitation of this study was the small sample size, reflecting its exploratory and preliminary nature (78).
Another limitation concerns the lack of accurate information on the diet of the dogs enrolled, given their aptitude for hunting and the consequent impossibility of dietary control. As such, the composition of the diet—particularly the exact concentration of FA—was not assessed. The inclusion of dietary data in future studies will be crucial, especially with regard to n-3 FA intake. However, we confirmed that none of the dogs received supplements highly enriched with these FA.
The use of whole blood as an analytical sample can also be considered a limitation, as plasma remains the most commonly used matrix (79). Nevertheless, this choice allowed us to explore a less conventional but potentially informative approach. Future studies using plasma will be important to determine whether lipid changes also occur at the cellular level (79). In addition, the erythrocyte membrane—known to reflect systemic health, nutritional, and metabolic status (32, 33, 77)—represents another promising sample type. In addition to plasma and erythrocytes, saliva should also be considered, as it has significant potential for periodontal research (128).
Finally, the limited number of studies investigating FA profiles in periodontal disease restricts the breadth of comparison and discussion of our results. These limitations can be overcome by expanding the sample size and incorporating lipidomic analyzes into future research. We also plan to include multiple dog breeds and apply advanced analytical techniques, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), which will allow for the evaluation of more complex lipid species, including oxylipids, ceramides, and phospholipids (129).
6 Conclusions
The findings indicate that alterations in whole-blood FA profiles reflect systemic inflammation associated with the progression of PD in dogs. Significant changes in specific FA and FA classes suggest a close link between lipid metabolism and PD pathogenesis. These FA may serve as promising biomarkers for early diagnosis and monitoring of canine periodontal disease.
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Ethics statement
The animal studies were approved by Ethics Committee of the University of Tre-os-Montes and Alto Douro—Comisss de issstes and Alto Douro Ethic-os-Montes e Alto Douro—Doc74-CE-UTAD-2024. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.
Author contributions
CS: Investigation, Writing – review & editing, Writing – original draft, Validation, Data curation, Methodology, Formal analysis. TS: Writing – review & editing, Methodology. MP: Methodology, Writing – review & editing. AF: Writing – original draft. AA: Writing – original draft. HB: Writing – review & editing. FP: Conceptualization, Software, Investigation, Funding acquisition, Writing – review & editing, Resources, Supervision, Project administration, Validation, Data curation, Methodology, Visualization, Formal analysis. RD: Project administration, Writing – review & editing, Supervision, Formal analysis, Validation, Methodology, Data curation, Visualization, Funding acquisition, Software, Conceptualization, Investigation, Resources. CV: Investigation, Conceptualization, Validation, Writing – review & editing, Supervision, Funding acquisition, Methodology, Software, Formal analysis, Project administration, Resources, Data curation, Visualization.
Funding
The author(s) declare that financial support was received for the research and/or publication of this article. The authors also acknowledge the financial support provided by FCT and the Ministry of Science and Technology (MCT) to the research units UID Centre for Environmental and Marine Studies (CESAM) + LA/P/0094/2020 and UID/50006 - Associated Laboratory of Green Chemistry - Clean Technologies and Processes. Nucleic Acids Res. This work was financially supported by the Vila Real Chemical Research Centre (CQ-VR) (UID/00616/2023). Foundation for Science and Technology, UI/00772, UIDB/04033/2020 and LA/P/0059/2020. The sponsors had no involvement in the work presented here.
Conflict of interest
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The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets.2025.1644675/full#supplementary-material
References
2. Wallis CV, Soltero-Rivera M, Harvey C, Reynolds RM, Carvell-Miller LJ, Colyer A, et al. Real-world diagnostic potential of bacterial biomarkers of canine periodontitis. Front Vet Sci. (2024) 11:1377119. doi: 10.3389/fvets.2024.1377119
3. American Veterinary Dental College. Veterinary dental nomenclature. J Vet Dent. (2007) 24, 54–7. doi: 10.1177/089875640702400111
4. Niemiec BA. Periodontal disease. Top Companion Anim Med. (2008) 23:72–80. doi: 10.1053/j.tcam.2008.02.003
5. Albuquerque C, Morinha F, Requicha J, Martins T, Dias I, Guedes-Pinto H, et al. Canine periodontitis: the dog as an important model for periodontal studies. Vet J. (2012) 191:299–305. doi: 10.1016/j.tvjl.2011.08.017
6. Silva C, Requicha J, Dias I, Bastos E, Viegas C. Genomic medicine in canine periodontal disease: a systematic review. Animals. (2023) 13:2463. doi: 10.3390/ani13152463
7. Wallis C, Saito EK, Salt C, Holcombe LJ, Desforges NG. Association of periodontal disease with breed size, breed, weight, and age in pure-bred client-owned dogs in the United States. Vet J. (2021) 275:105717. doi: 10.1016/j.tvjl.2021.105717
8. Wallis C, Holcombe LJA. Review of the frequency and impact of periodontal disease in dogs. J Small Anim Pract. (2020) 61:529–40. doi: 10.1111/jsap.13218
9. Whyte A, Whyte J, Monteagudo LV, García-Barrios A, Teresa Tejedor M. Periodontal and dental status in packs of Spanish dogs. Animals. (2021) 11:1082. doi: 10.3390/ani11041082
10. Harvey CE. Management of periodontal disease: understanding the options. Vet Clin North Am Small Anim Pract. (2005) 35:819–36. doi: 10.1016/j.cvsm.2005.03.002
11. Enlund KB, Jönsson B, Abrahamsson KH, Pettersson A. Long-term effects of motivational interviewing vs. traditional counseling on dog owners' adherence to veterinary dental home care: a three-year follow-up study. Front Vet Sci. (2024) 11:1296618. doi: 10.3389/fvets.2024.1296618
12. Lourenço AL, Booij-Vrieling HE, Vossebeld CB, Neves A, Viegas C, Corbee RJ, et al. The effect of dietary corn oil and fish oil supplementation in dogs with naturally occurring gingivitis. J Anim Physiol Anim Nutr. (2018) 102:1382–9. doi: 10.1111/jpn.12932
13. Bartold PM, Walsh LJ, Narayanan AS. Molecular and cell biology of the gingiva. Periodontol 2000 24. (2000) 28–55. doi: 10.1034/j.1600-0757.2000.2240103.x
14. Page RC, Offenbacher S, Schroeder HE, Seymour GJ, Kornman KS. Advances in the pathogenesis of periodontitis: summary of developments, clinical implications and future directions. Periodontol 2000. (1997) 14:216–48. doi: 10.1111/j.1600-0757.1997.tb00199.x
15. Nussbaum G, Shapira L. How has neutrophil research improved our understanding of periodontal pathogenesis? J Clin Periodontol. (2011) 38:49–59. doi: 10.1111/j.1600-051X.2010.01678.x
16. Sorsa T, Tjäderhane L, Konttinen YT, Lauhio A, Salo T, Lee HM, et al. Matrix metalloproteinases: contribution to pathogenesis, diagnosis and treatment of periodontal inflammation. Ann Med. (2006) 38:306–21. doi: 10.1080/07853890600800103
17. Chee B, Park B, Fitzsimmons T, Coates AM, Bartold PM. Omega-3 fatty acids as an adjunct for periodontal therapy—a review. Clin Oral Invest. (2016) 20:879–94. doi: 10.1007/s00784-016-1750-2
18. Sun D, Krishnan A, Zaman K, Lawrence R, Bhattacharya A, Fernandes G, et al. Dietary N-3 fatty acids decrease osteoclastogenesis and loss of bone mass in ovariectomized mice. J Bone Miner Res. (2003) 18:1206–16. doi: 10.1359/jbmr.2003.18.7.1206
19. Döding A, Zimmermann S, Maghames A, Reimann M, Symmank J, Thürmer M, et al. Immunometabolic capacities of nutritional fatty acids in regulation of inflammatory bone cell interaction and systemic impact of periodontal infection. Front Immunol. (2023) 14:1213026. doi: 10.3389/fimmu.2023.1213026
20. Martínez-García M, Hernández-Lemus E. Periodontal inflammation and systemic diseases: an overview. Front Physiol. (2021) 12:709438. doi: 10.3389/fphys.2021.709438
21. Wang CW, McCauley LK. Osteoporosis and periodontitis. Curr Osteoporos Rep. (2016) 14:284–91. doi: 10.1007/s11914-016-0330-3
22. Morinha F, Albuquerque C, Requicha J, Dias I, Leitão J, Gut I, et al. Detection and characterization of interleukin-6 gene variants in canis familiaris: association studies with periodontal disease. Gene. (2011) 485:139–45. doi: 10.1016/j.gene.2011.06.018
23. Tamura K, Tokuzen-Tai M, Siddiqui YD, Tamura-Naito H, Nagahara Y, Hatanaka-Takeuchi K, et al. Estimation of periodontal pocket surface area in small to medium dogs: a proof-of-concept study. BMC Vet Res. (2022) 18:13. doi: 10.1186/s12917-021-03116-0
24. Kassebaum NJ, Bernabé E, Dahiya M, Bhandari B, Murray CJL, Marcenes W, et al. Global burden of severe periodontitis in 1990-2010: a systematic review and meta-regression. J Dent Res. (2014) 93:1045–53. doi: 10.1177/0022034514552491
25. Li T, Wu H, Fu Z, Li H, Li Q, Liu Y, et al. The association between polyunsaturated fatty acids and periodontitis: NHANES 2011–2014 and mendelian randomisation analysis. Lipids Health Dis. (2024) 23:168. doi: 10.1186/s12944-024-02159-0
26. Harvey CE. Periodontal disease in dogs. Etiopathogenesis, prevalence, and significance. Vet Clin North Am Small Anim Pract. (1998) 28:1111–28. doi: 10.1016/S0195-5616(98)50105-2
27. Silva C, Abrantes AC, Fontes AC, Dias I, Domingues R, Peixoto F, et al. Evaluation of haematological ratios at: different stages of canine periodontal disease. Vet Sci. (2024) 11:581. doi: 10.3390/vetsci11110581
28. Züllig T, Trötzmüller M, Köfeler HC. Lipidomics from sample preparation to data analysis: a primer. Anal Bioanal Chem. (2020) 412:2191–209. doi: 10.1007/s00216-019-02241-y
29. Calder PC. The relationship between the fatty acid composition of immune cells and their function. Prostaglandins Leukot Essent Fatty Acids. (2008) 79:101–8. doi: 10.1016/j.plefa.2008.09.016
30. Calder PC. Marine omega-3 fatty acids and inflammatory processes: effects, mechanisms and clinical relevance. Biochim Biophys Acta. (2015) 1851:469–84. doi: 10.1016/j.bbalip.2014.08.010
31. Ma C, Vasu R, Zhang H. The role of long-chain fatty acids in inflammatory bowel disease. Mediators Inflamm. (2019) 2019:8495913. doi: 10.1155/2019/8495913
32. Crisi PE, Giordano MV, Luciani A, Gramenzi A, Prasinou P, Sansone A, et al. Evaluation of the fatty acid-based erythrocyte membrane lipidome in cats with food responsive enteropathy, inflammatory bowel disease and low-grade intestinal T-cell lymphoma. PLoS ONE. (2024) 19:e0307757. doi: 10.1371/journal.pone.0307757
33. Crisi PE, Luciani A, Di Tommaso M, Prasinou P, De Santis F, Chatgilialoglu MV, et al. The fatty acid-based erythrocyte membrane lipidome in dogs with chronic enteropathy. Animals. (2021) 11:2604. doi: 10.3390/ani11092604
34. Huang Y, Zhu M, Li Z, Sa R, Chu Q, Zhang Q, et al. Mass spectrometry-based metabolomic profiling identifies alterations in salivary redox status and fatty acid metabolism in response to inflammation and oxidative stress in periodontal disease. Free Radic Biol Med. (2014) 70:223–32. doi: 10.1016/j.freeradbiomed.2014.02.024
35. Freeman LM, Rush JE, Kehayias JJ, Ross JN, Meydani SN, Brown DJ, et al. Nutritional alterations and the effect of fish oil supplementation in dogs with heart failure. J Vet Intern Med. (1998) 12:440–8. doi: 10.1111/j.1939-1676.1998.tb02148.x
36. Mueller RS, Fieseler KV, Rosychuk RAW, Ogilvie GK, Greenwalt TL. Effect of omega-3 fatty acids on canine atopic dermatitis. J Small Anim Pract. (2004) 45:293–7. doi: 10.1111/j.1748-5827.2004.tb00238.x
37. Logas D, Kunkle GA. Double-blinded crossover study with marine oil supplementation containing high-dose eicosapentaenoic acid for the treatment of canine pruritic skin disease. Vet Dermatol. (1994) 5:99–104. doi: 10.1111/j.1365-3164.1994.tb00020.x
38. Abba C, Mussa PP, Vercelli A, Raviri G. Essential fatty acids supplementation in different-stage atopic dogs fed on a controlled diet. J Anim Physiol Anim Nutr. (2005) 89:203–7. doi: 10.1111/j.1439-0396.2005.00541.x
39. Brown SA. Oxidative stress and chronic kidney disease. Vet Clin Small Anim. (2008) 38:157–66. doi: 10.1016/j.cvsm.2007.11.001
40. Brown SA, Brown CA, Crowell WA, Barsanti JA, Allen T, Cowell C, et al. Beneficial effects of chronic administration of dietary ω-3 polyunsaturated fatty acids in dogs with renal insufficiency. J Lab Clin Med. (1998) 131:447–55. doi: 10.1016/S0022-2143(98)90146-9
41. Brown SA, Brown CA, Crowell WA, Barsanti JA, Kang CW, Allen T, et al. Effects of dietary polyunsaturated fatty acid supplementation in early renal insufficiency in dogs. J Lab Clin Med. (2000) 135:275–86. doi: 10.1067/mlc.2000.105178
42. Roush JK, Cross AR, Renberg WC, Dodd CE, Sixby KA, Fritsch DA, et al. Evaluation of the effects of dietary supplementation with fish oil omega-3 fatty acids on weight bearing in dogs with osteoarthritis. J Am Vet Med Assoc. (2010) 236:67–73. doi: 10.2460/javma.236.1.67
43. Hansen RA, Harris MA, Pluhar GE, Motta T, Brevard S, Ogilvie GK, et al. Fish oil decreases matrix metalloproteinases in knee synovia of dogs with inflammatory joint disease. J Nutr Biochem. (2008) 19:101–8. doi: 10.1016/j.jnutbio.2007.01.008
44. Fritsch D, Allen TA, Dodd CE, Jewell DE, Sixby KA, Leventhal PS, et al. Dose-titration effects of fish oil in osteoarthritic dogs. J Vet Intern Med. (2010) 24:1020–6. doi: 10.1111/j.1939-1676.2010.0572.x
45. Roush JK, Dodd CE, Fritsch DA, Allen TA, Jewell DE, Schoenherr WD, et al. Multicenter veterinary practice assessment of the effects of omega-3 fatty acids on osteoarthritis in dogs. J Am Vet Med Assoc. (2010) 236:59–66. doi: 10.2460/javma.236.1.59
46. Browning LM, Walker CG, Mander AP, West AL, Madden J, Gambell JM, et al. Incorporation of eicosapentaenoic and docosahexaenoic acids into lipid pools when given as supplements providing doses equivalent to typical intakes of oily fish. Am J Clin Nutr. (2012) 96:748–58. doi: 10.3945/ajcn.112.041343
47. Silva V, Barazzoni R, Singer P. Biomarkers of fish oil omega-3 polyunsaturated fatty acids intake in humans. Nutr Clin Pract. (2014) 29:63–72. doi: 10.1177/0884533613516144
48. Cleland LG, James MJ, Proudman SM. Fish oil: what the prescriber needs to know. Arthritis Res Ther. (2006) 8:202. doi: 10.1186/ar1876
49. Rahman MM, Bhattacharya A, Fernandes G. Docosahexaenoic acid is more potent inhibitor of osteoclast differentiation in RAW 264.7 cells than eicosapentaenoic acid. J Cell Physiol. (2008) 214:201–9. doi: 10.1002/jcp.21188
50. Peterson LD, Jeffery NM, Thies F, Sanderson P, Newsholme EA, Calder PC, et al. Eicosapentaenoic and docosahexaenoic acids alter rat spleen leukocyte fatty acid composition and prostaglandin E2 production but have different effects on lymphocyte functions and cell-mediated immunity. Lipids. (1998) 33:171–80. doi: 10.1007/s11745-998-0193-y
51. Chapkin RS, Akoh CC, Miller CC. Influence of dietary N-3 fatty acids on macrophage glycerophospholipid molecular species and peptidoleukotriene synthesis. J Lipid Res. (1991) 32:1205–13. doi: 10.1016/S0022-2275(20)41983-2
52. Yaqoob P, Calder P. Effects of dietary lipid manipulation upon inflammatory mediator production by murine macrophages. Cell Immunol. (1995) 163:120–8. doi: 10.1006/cimm.1995.1106
53. Sierra S, Lara-Villoslada F, Comalada M, Olivares M, Xaus J. Dietary eicosapentaenoic acid and docosahexaenoic acid equally incorporate as decosahexaenoic acid but differ in inflammatory effects. Nutrition. (2008) 24:245–54. doi: 10.1016/j.nut.2007.11.005
54. Chiang N, Serha CN. Specialized pro-resolving mediator network: an update on production and actions. Essays Biochem. (2020) 64:443–62. doi: 10.1042/EBC20200018
55. Bartha V, Exner L, Basrai M, Bischoff SC, Schweikert D, Adolph M, et al. Changes in serum omega fatty acids on a mediterranean diet intervention in patients with gingivitis: an exploratory study. J Periodontal Res. (2022) 57:1198–209. doi: 10.1111/jre.13056
56. Serhan CN, Yacoubian S, Yang R. Anti-inflammatory and proresolving lipid mediators. Annu Rev Pathol. (2008) 3:279–312. doi: 10.1146/annurev.pathmechdis.3.121806.151409
57. Carlucci AR, Bergo BR, Silva RN de B, Bressane G de D, Baeza M, Santos NC, et al. Dos effects of host modulation through omega-3 dietary supplementation on inflammatory outcomes in periodontitis: a scoping review. Einstein. (2024) 22:eRW0936. doi: 10.31744/einstein_journal/2024RW0936
58. Hajishengallis G, Chavakis T, Lambris JD. Current understanding of periodontal disease pathogenesis and targets for host-modulation therapy. Periodontol 2000. (2020) 84:14–34. doi: 10.1111/prd.12331
59. Calder PC. Omega-3 fatty acids and inflammatory processes. Nutrients. (2010) 2:355–74. doi: 10.3390/nu2030355
60. Balta MG, Papathanasiou E, Blix IJ, Van Dyke TE. Host modulation and treatment of periodontal disease. J Dent Res. (2021) 100:798–809. doi: 10.1177/0022034521995157
61. Eltay EG, Van Dyke T. Resolution of inflammation in oral diseases. Pharmacol Ther. (2023) 247:108453. doi: 10.1016/j.pharmthera.2023.108453
62. Thornton JM, Yin K. Role of specialized pro-resolving mediators in modifying host defense and decreasing bacterial virulence. Molecules. (2021) 26:6970. doi: 10.3390/molecules26226970
63. Kroetz DL, Zeldin DC, Williams L. Cytochrome P450 pathways of arachidonic acid metabolism. Curr Opin Lipidol. (2002) 13:273–83. doi: 10.1097/00041433-200206000-00007
64. Lewis RA, Austen KF, Soberman RJ. Leukotrienes and other products of the 5-lipoxygenase pathway. N Engl J Med. (1990) 323:645–55. doi: 10.1056/NEJM199009063231006
65. Tilley SL, Coffman TM, Koller BH. Mixed messages: modulation of inflammation and immune responses by prostaglandins and thromboxanes. J Clin Invest. (2001) 108:15–23. doi: 10.1172/JCI200113416
66. Simopoulos AP. Importance of the omega-6/omega-3 balance in health and disease: evolutionary aspects of diet. World Rev Nutr Diet. (2011) 102:10–21. doi: 10.1159/000327785
67. Ashrafzadeh E, Babaei H, Ravanbakhsh M, Zare Javid A, Maghsoumi-Norouzabad L. Beneficial effects of cranberry juice enriched with omega-3 fatty acids in patients with type 2 diabetic and periodontal disease: a randomized pilot clinical trial. J Adv Periodontol Implant Dent. (2024) 16:160–72. doi: 10.34172/japid.2024.019
68. Choi J-S, Park N-H, Hwang S-Y, Sohn JH, Kwak I, Cho KK, et al. The antibacterial activity of various saturated and unsaturated fatty acids against several oral pathogens. J Environ Biol. (2013) 34:673–6.
69. Araghizadeh N, Paknejad M, Alaeddini M, Minaii B, Abdollahi M, Khorasanie R, et al. The efficacy and prophylactic characteristics of omega-3 fatty acids in experimental gingivitis in rats. Iran J Basic Med Sci. (2014) 17:87–92.
70. Bendyk A, Marino V, Zilm PS, Howe P, Bartold PM. Effect of dietary omega-3 polyunsaturated fatty acids on experimental periodontitis in the mouse. J Periodont Res. (2009) 44:211–6. doi: 10.1111/j.1600-0765.2008.01108.x
71. Hasturk H, Kantarci A, Goguet-Surmenian E, Blackwood A, Andry C, Serhan CN, et al. Resolvin E1 regulates inflammation at the cellular and tissue level and restores tissue homeostasis in vivo. J Immunol. (2007) 179:7021–9. doi: 10.4049/jimmunol.179.10.7021
72. Schwab JM, Chiang N, Arita M, Serhan CN. Resolvin E1 and Protectin D1 activate inflammation-resolution programmes. Nature. (2007) 447:869–74. doi: 10.1038/nature05877
73. Hall DJ, Freeman LM, Rush JE, Cunningham SM. Comparison of serum fatty acid concentrations in cats with hypertrophic cardiomyopathy and healthy controls. J Feline Med Surg. (2014) 16:631–6. doi: 10.1177/1098612X13516478
74. Stańdo-Retecka M, Piatek P, Namiecinska M, Bonikowski R, Lewkowicz P, Lewkowicz N, et al. Clinical and microbiological outcomes of subgingival instrumentation supplemented with high-dose omega-3 polyunsaturated fatty acids in periodontal treatment—a randomized clinical trial. BMC Oral Health. (2023) 23:290. doi: 10.1186/s12903-023-03018-7
75. Kesavalu L, Bakthavatchalu V, Rahman MM, Su J, Raghu B, Dawnson D, et al. Omega-3 fatty acid regulates cytokine/mediator rna expression in porphyromonas gingivalis - induced experimental periodontal. Oral Microbiol Immunol. (2007) 22:232–9. doi: 10.1111/j.1399-302X.2007.00346.x
76. Sieber-Ruckstuhl NS, Tham WK, Baumgartner F, Selva JJ, Wenk MR, Burla B, et al. Serum lipidome signatures of dogs with different endocrinopathies associated with hyperlipidemia. Metabolites. (2022) 12:306. doi: 10.3390/metabo12040306
77. Prasinou P, Crisi PE, Chatgilialoglu C, Tommaso Di, Sansone M, Gramenzi A, et al. Lipidome of healthy dogs: creating a benchmark of fatty acid distribution and interval values. Front Vet Sci. (2020) 7:502. doi: 10.3389/fvets.2020.00502
78. Boretti FS, Burla B, Deuel J, Gao L, Wenk MR, Liesegang A, et al. Serum lipidome analysis of healthy beagle dogs receiving different diets. Metabolomics. (2020) 16:1. doi: 10.1007/s11306-019-1621-3
79. Ferreira HB, Melo T, Guerra IMS, Moreira ASP, Laranjeira P, Paiva A, et al. Whole blood and plasma-based lipid profiling reveals distinctive metabolic changes in systemic lupus erythematosus and systemic sclerosis. J Proteome Res. (2023) 22:2995–3008. doi: 10.1021/acs.jproteome.3c00321
80. du Sert NP, Ahluwalia A, Alam S, Avey MT, Baker M, Browne WJ, et al. Reporting animal research: explanation and elaboration for the ARRIVE guidelines 2.0. PLoS Biol. (2020) 18:e3000411. doi: 10.1371/journal.pbio.3000411
81. Bligh EG, Dyer WJA. Rapid method of total lipid extraction and purification. Can J Biochem Physiol. (1959) 37:911–7. doi: 10.1139/o59-099
82. Monteiro-Cardoso VF, Castro M, Oliveira MM, Moreira PI, Peixoto F, Videira RA, et al. Age-dependent biochemical dysfunction in skeletal muscle of triple-transgenic mouse model of Alzheimer's disease. Curr Alzheimer Res. (2015) 12:100–15. doi: 10.2174/1567205012666150204124852
83. Peştean CP, Pocquet H, Dumitraş DA, Morohoschi AG, Ştefǎnuţ. rohosc LC, Andrei S, et al. Correlation between oxidative stress markers and periodontal disease in dogs. Vet Sci. (2024) 11:99. doi: 10.3390/vetsci11030099
84. Sculley DV, Langley-Evans SC. Periodontal disease is associated with lower antioxidant capacity in whole saliva and evidence of increased protein oxidation. Clin Sci. (2003) 105:167–72. doi: 10.1042/CS20030031
85. Mirnic J, Djuric M, Brkic S, Gusic I, Stojilkovic M, Tadic A, et al. Pathogenic mechanisms that may link periodontal disease and type 2 diabetes mellitus—the role of oxidative stress. Int J Mol Sci. (2024) 25:9806. doi: 10.3390/ijms25189806
86. Chen X, He Y, Zhou Y, Gong H, Zhang J, Qiu G, et al. Modulatory role of exogenous arachidonic acid in periodontitis with type 2 diabetes mellitus mice. BMC Oral Health. (2025) 25:264. doi: 10.1186/s12903-025-05525-1
87. Harvey CE, Shofer FS, Laster L. Association of age and body weight with periodontal disease in North American dogs. J Vet Dent. (1994) 11:94–105. doi: 10.1177/089875649401100301
88. Pavlica Z, Petelin M, Nemec A, Erzen D, Skaleric U. Measurement of total antioxidant capacity in gingival crevicular fluid and serum in dogs with periodontal disease. Am J Vet Res. (2004) 65:1584–8. doi: 10.2460/ajvr.2004.65.1584
89. Lepsch J, Farias DR, Eshriqui I, Rebelo F, dos Santos Vaz J, Adegboye AA, et al. Serum fatty acids are positively associated with changes in systemic blood pressure throughout pregnancy. Pregnancy Hypertens. (2018) 13:7–13. doi: 10.1016/j.preghy.2018.04.012
90. Arghavani H, Bilodeau JF, Rudkowska I. Association between circulating fatty acids and blood pressure: a review. Curr Nutr Rep. (2025) 14:15. doi: 10.1007/s13668-024-00602-3
91. Hujoel PP, Lingström P. Nutrition, dental caries and periodontal disease: a narrative review. J Clin Periodontol. (2017) 44:S79–84. doi: 10.1111/jcpe.12672
92. Kyllar M, Witter K. Prevalence of dental disorders in pet dogs. Vet Med. (2005) 2005:496–505. doi: 10.17221/5654-VETMED
93. Drozd A, Kotlega D, Nowacki P, Ciećwież S, Trochanowski T, Szczuko M, et al. Fatty acid levels and their inflammatory metabolites are associated with the nondipping status and risk of obstructive sleep apnea syndrome in stroke patients. Biomedicines. (2022) 10:2200. doi: 10.3390/biomedicines10092200
94. Li X, Huang Y, Zhang W, Yang C, Su W, Wu Y, et al. Association of circulating saturated fatty acids with the risk of pregnancy-induced hypertension: a nested case–control study. Hypertens Res. (2020) 43:412–21. doi: 10.1038/s41440-019-0383-7
95. Morandi A, Piona C, Bonafini S, Marigliano M, Tomasselli F, Tagetti A, et al. Long chain fatty acids metabolism and cardiovascular risk factors in youth with type 1 diabetes. Nutr Metab Cardiovasc Dis. (2021) 31:297–305. doi: 10.1016/j.numecd.2020.08.023
96. Yang B, Ding F, Yan J, Ye XW, Xu XL, Wang FL, et al. Exploratory serum fatty acid patterns associated with blood pressure in community-dwelling middle-aged and elderly Chinese. Lipids Health Dis. (2016) 15:58. doi: 10.1186/s12944-016-0226-3
97. Figueredo CM, Martinez GL, Koury JC, Fischer RG, Gustafsson A. Serum levels of long-chain polyunsaturated fatty acids in patients with periodontal disease. J Periodontol. (2013) 84:675–82. doi: 10.1902/jop.2012.120171
98. Requirand P, Gibert P, Tramini P, Cristol JP, Descomps B. Serum fatty acid imbalance in bone loss: example with periodontal disease. Clin Nutr. (2000) 19:271–6. doi: 10.1054/clnu.2000.0107
99. Ramirez-Tortosa MC, Quiles JL, Battino M, Granados S, Morillo JM, Bompadre S, et al. Periodontitis is associated with altered plasma fatty acids and cardiovascular risk markers. Nutr Metab Cardiovasc Dis. (2010) 20:133–9. doi: 10.1016/j.numecd.2009.03.003
100. Kantarci A, Van Dyke TE. Lipoxin signaling in neutrophils and their role in periodontal disease. Prostaglandins Leukot Essent Fatty Acids. (2005) 73:289–99. doi: 10.1016/j.plefa.2005.05.019
101. Yin H, Zhou Y, Zhu M, Hou S, Li Z, Zhong H, et al. Role of mitochondria in programmed cell death mediated by arachidonic acid-derived eicosanoids. Mitochondrion. (2013) 13:209–24. doi: 10.1016/j.mito.2012.10.003
102. Funk CD. Prostaglandins and leukotrienes: advances in eicosanoid biology. Science (1979). (2001) 294:1871–5. doi: 10.1126/science.294.5548.1871
103. Calder PC. N-3 polyunsaturated fatty acids and inflammation: from molecular biology to the clinic. Lipids. (2003) 38:343–52. doi: 10.1007/s11745-003-1068-y
104. Fredman G, Oh SF, Ayilavarapu S, Hasturk H, Serhan CN, van Dyke TE, et al. Impaired phagocytosis in localized aggressive periodontitis: rescue by resolvin E1. PLoS ONE. (2011) 6:e24422. doi: 10.1371/journal.pone.0024422
105. Yin H, Xu L, Porter NA. Free radical lipid peroxidation: mechanisms and analysis. Chem Rev. (2011) 111:5944–72. doi: 10.1021/cr200084z
106. Shen Z, Ma Y, Ji Z, Hao Y, Yan X, Zhong Y, et al. Arachidonic acid induces macrophage cell cycle arrest through the JNK signaling pathway. Lipids Health Dis. (2018) 17:26. doi: 10.1186/s12944-018-0673-0
107. Das UN. Arachidonic acid and other unsaturated fatty acids and some of their metabolites function as endogenous antimicrobial molecules: a review. J Adv Res. (2018) 11:57–66. doi: 10.1016/j.jare.2018.01.001
108. Pereira dos Santos JD, Cunha E, Nunes T, Tavares L, Oliveira M. Relation between Periodontal Disease and Systemic Diseases in Dogs. Res Vet Sci. (2019) 125:136–40. doi: 10.1016/j.rvsc.2019.06.007
109. Glickman LT, Glickman NW, Moore GE, Goldstein GS, Lewis HB. Evaluation of the risk of endocarditis and other cardiovascular events on the basis of the severity of periodontal disease in dogs. J Am Vet Med Assoc. (2009) 234:486–94. doi: 10.2460/javma.234.4.486
110. Zhou Y, Khan H, Xiao J, Cheang WS. Effects of arachidonic acid metabolites on cardiovascular health and disease. Int J Mol Sci. (2021) 22:12029. doi: 10.3390/ijms222112029
111. Basil MC, Levy BD. Specialized pro-resolving mediators: endogenous regulators of infection and inflammation. Nat Rev Immunol. (2016) 16:51–67. doi: 10.1038/nri.2015.4
112. Serhan CN, Chiang N, Dalli J. The resolution code of acute inflammation: novel pro-resolving lipid mediators in resolution. Semin Immunol. (2015) 27:200–15. doi: 10.1016/j.smim.2015.03.004
113. Zhang Y, Chen H, Zhang W, Cai Y, Shan P, Wu D, et al. Arachidonic acid inhibits inflammatory responses by binding to myeloid differentiation factor-2 (MD2) and preventing MD2/toll-like receptor 4 signaling activation. Biochim Biophys Acta Mol Basis Dis. (2020) 1866:165683. doi: 10.1016/j.bbadis.2020.165683
114. Sergeant S, Rahbar E, Chilton FH. Gamma-linolenic acid, dihommo-gamma linolenic, eicosanoids and inflammatory processes. Eur J Pharmacol. (2016) 785:77–86. doi: 10.1016/j.ejphar.2016.04.020
115. Burr GO, Burr MMA. New deficiency disease produced by the rigid exclusion of fat from the diet. J Biol Chem. (1929) 82:345–67. doi: 10.1016/S0021-9258(20)78281-5
116. Richter CK, Bisselou KS, Nordgren TM, Smith L, Appiah AK, Hein N, et al. N-3 docosapentaenoic acid intake and relationship with plasma long-chain n-3 fatty acid concentrations in the United States: NHANES 2003–2014. Lipids. (2019) 54:221–30. doi: 10.1002/lipd.12146
117. Julliard WA, Myo YPA, Perelas A, Jackson PD, Thatcher TH, Sime PJ, et al. Specialized pro-resolving mediators as modulators of immune responses. Semin Immunol. (2022) 59:101605. doi: 10.1016/j.smim.2022.101605
118. Skulas-Ray AC, Flock MR, Richter CK, Harris WS, West SG, Kris-Etherton PM, et al. Red Blood cell docosapentaenoic acid (DPA n-3) is inversely associated with triglycerides and C-Reactive Protein (CRP) in healthy adults and dose-dependently increases following n-3 fatty acid supplementation. Nutrients. (2015) 7:6390–404. doi: 10.3390/nu7085291
119. Hwang DH, Kim JA, Lee JY. Mechanisms for the activation of toll-like receptor 2/4 by saturated fatty acids and inhibition by docosahexaenoic acid. Eur J Pharmacol. (2016) 785:24–35. doi: 10.1016/j.ejphar.2016.04.024
120. Lemaitre RN, Tanaka T, Tang W, Manichaikul A, Foy M, Kabagambe EK, et al. Genetic loci associated with plasma phospholipid N-3 fatty acids: a meta-analysis of genome-wide association studies from the charge consortium. PLoS Genet. (2011) 7:e1002193. doi: 10.1371/journal.pgen.1002193
121. Sanders AE, Weatherspoon ED, Ehrmann BM, Soma PS, Shaikh SR, Preisser JS, et al. Ratio of omega-6/omega-3 polyunsaturated fatty acids associated with somatic and depressive symptoms in people with painful temporomandibular disorder and irritable bowel syndrome. J Pain. (2022) 23:1737–48. doi: 10.1016/j.jpain.2022.04.006
122. Hellström A, Hellström W, Hellgren G, Smith LEH, Puttonen H, Fyhr IM, et al. Docosahexaenoic acid and arachidonic acid levels are associated with early systemic inflammation in extremely preterm infants. Nutrients. (2020) 12:1996. doi: 10.3390/nu12071996
123. Alashmali SM, Lin L, Trépanier MO, Cisbani G, Bazinet RP. The effects of N-6 polyunsaturated fatty acid deprivation on the inflammatory gene response to lipopolysaccharide in the mouse hippocampus. J Neuroinflammation. (2019) 16:237. doi: 10.1186/s12974-019-1615-0
124. Sztolsztener K, Chabowski A, Harasim-Symbor E, Bielawiec P, Konstantynowicz-Nowicka K. Arachidonic acid as an early indicator of inflammation during non-alcoholic fatty liver disease development. Biomolecules. (2020) 10:1133. doi: 10.3390/biom10081133
125. Larsson SC, Carter P, Vithayathil M, Mason AM, Michaëlsson K, Baron JA, et al. Genetically predicted plasma phospholipid arachidonic acid concentrations and 10 site-specific cancers in UK Biobank and genetic consortia participants: a mendelian randomization study. Clin Nutr. (2021) 40:3332–7. doi: 10.1016/j.clnu.2020.11.004
126. Pouliot M, Clish CB, Petasis NA, Van Dyke TE, Serhan CN. Lipoxin A4 analogues inhibit leukocyte recruitment to porphyromonas gingivalis: a role for cyclooxygenase-2 and lipoxins in periodontal disease. Biochemistry. (2000) 39:4761–8. doi: 10.1021/bi992551b
127. Holmstrom SE, Bellows J, Juriga S, Knutson K, Niemiec BA, Perrone J, et al. 2013 AAHA dental care guidelines for dogs and cats*. J Am Anim Hosp Assoc. (2013) 49:75–82. doi: 10.5326/JAAHA-MS-4013
128. Silva C, Peixoto F, Dias I, Domingues R, Viegas C. Lipidomics in periodontal disease research: a systematic review. Curr Med Chem. (2024) 32:3825–49 doi: 10.2174/0109298673323591240923051742
Keywords: dog, fatty acids, gingivitis, lipidomics, periodontal disease, periodontitis, whole blood
Citation: Silva C, Sousa T, Pinho M, Fontes AC, Abrantes AC, Brancal H, Peixoto F, Domingues R and Viegas C (2025) Plasticity of the fatty acid whole blood lipidome in the progression of canine periodontal disease: a pilot study. Front. Vet. Sci. 12:1644675. doi: 10.3389/fvets.2025.1644675
Received: 10 June 2025; Revised: 13 November 2025;
Accepted: 27 November 2025; Published: 18 December 2025.
Edited by:
Damián Escribano, University of Murcia, SpainReviewed by:
Ravikanthreddy Poonooru, University of Missouri, United StatesShogo Takashiba, Okayama University, Japan
Copyright © 2025 Silva, Sousa, Pinho, Fontes, Abrantes, Brancal, Peixoto, Domingues and Viegas. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Carlos Viegas, Y3ZpZWdhc0B1dGFkLnB0