# Dietary pentadecanoic acid supplementation at weaning in essential fatty acid-deficient rats shed light on the new family of odd-chain n-8 PUFAs

Vincent Ciesielski, Thomas Guerbette, Léa Fret, Mélodie Succar, Youenn

Launay, Patrice Dahirel, Philippe P. Legrand, Manuel Vlach, Sophie Blat, Vincent V. Rioux

<figure><img src="image_1.png" /></figure>

## To cite this version:

Vincent Ciesielski, Thomas Guerbette, Léa Fret, Mélodie Succar, Youenn Launay, et al. Dietary pentadecanoic acid supplementation at weaning in essential fatty acid-deficient rats shed light on the new family of odd-chain n-8 PUFAs. Journal of Nutritional Biochemistry, 2025, 137, pp.109814. 10.1016/j.jnutbio.2024.109814. hal-04831868

HAL Id: hal-04831868

https://hal.inrae.fr/hal-04831868v1

Submitted on 16 May 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
RESEARCH PAPER

Dietary pentadecanoic acid supplementation at weaning in essential fatty acid-deficient rats shed light on the new family of odd-chain n-8 PUFAs

Vincent Ciesielski<sup>a,b</sup>, Thomas Guerbette<sup>a,b</sup>, Léa Fret<sup>a</sup>, Mélodie Succar<sup>a</sup>, Youenn Launay<sup>a,b</sup>, Patrice Dahirel<sup>b</sup>, Philippe Legrand<sup>a,b</sup>, Manuel Vlach<sup>a,b</sup>, Sophie Blat<sup>b</sup>, Vincent Rioux<sup>a,b,*</sup>

Received 10 May 2024; received in revised form 22 November 2024; accepted 27 November 2024

Abstract

Pentadecanoic acid (C15:0) is a saturated odd-chain fatty acid (OCFA), mainly found in dairy products. Its physiological and nutritional effects are still unknown, yet some recent evidences suggest it might be beneficial to human health. Moreover, pentadecanoic acid has recently been suspected of having essential roles in humans, although the mechanisms are not described. We therefore questioned the potential essentiality of this fatty acid (FA). We investigated *in vivo* the effect of a C15:0 supplementation on essential fatty acid (EFA) deficient Wistar rats. Female rats were fed an EFA-deficient diet 2 weeks before mating, during pregnancy and lactation. Weaned pups were fed the EFA-deficient diet or were switched to a diet supplemented with C15:0 or linoleic acid (LA) for 11 weeks. A control group was fed with EFA during the whole study. Since linoleic acid deficiency is known to induce growth delay, weights were measured throughout the experiment and FA content in collected tissues were analyzed to evaluate biochemical markers of the deficiency. As expected, EFA-deficient rats showed growth retardation, compared to control rats. Supplementation of C15:0 at weaning increased early growth rate compared to deficient animals, as also did the supplementation of C18:2 n-6. Furthermore, the supplementation of C15:0 in the diet of EFA-deficient animals induced the previously undescribed synthesis of odd-chain PUFAs of the n-8 family (C19:3, C21:3 and C21:4 n-8). These results suggest dietary C15:0 might counteract EFA induced growth retardation, possibly through the synthesis of odd-chain n-8 PUFAs, yet mechanisms are to be deciphered for further validation.

© 2024 The Author(s). Published by Elsevier Inc.

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Keywords: Essential fatty acids; Dietary pentadecanoic acid; Odd chain fatty acids; Essential fatty acid deficiency; n-8 odd-chain PUFAs.

1. Introduction

Since the mid-20th century, saturated fatty acids (SFAs) were
often associated with detrimental health issues, leading to a re-
striction of their consumption in most dietary guidelines. Indeed,
early observational studies suggested that high consumption of
SFAs increased plasma low-density lipoprotein (LDL)-cholesterol
and was associated with elevated risk of cardiovascular disease
(CVD) [1]. Yet, recent meta-analyses showed there is no significant
evidence of the association between SFAs and CVD [2]. In addition

to this controversial association, studies now suggest SFAs should
not be considered as a single group due to different properties of
individual SFA [3].

The great majority of studies about dietary SFAs concern SFAs
with even number of carbon atoms, which are the predominant
natural forms of saturated fat. SFAs with an odd number of carbon
(Odd Chain Fatty Acids, OCFAs), like pentadecanoic acid (C15:0)
or heptadecanoic acid (C17:0), are minor forms of SFAs, described
mainly in ruminant milk and dairy products. Linear OCFAs are in-
deed formed when propionyl-CoA is used instead of acetyl-CoA as

Abbreviations: ALA, Alpha linolenic acid; ARA, Arachidonic acid; C15:0, Pentadecanoic acid; C17:0, Heptadecanoic acid; CVD, cardiovascular disease; DHA, Docosahexaenoic acid; EFA, Essential fatty acid; FA, Fatty acid; FAEE, Fatty acid ethyl ester; FAME, Fatty acid methyl ester; GC-MS, gas chromatography-mass spectrometry; IGF-1, Insulin growth factor 1; LA, linoleic acid; LDL, Low-density lipoprotein; MA, Mead acid; NASH, Non-alcoholic steatohepatitis; OCFA, Odd chain fatty acid; OCPUFA, odd chain polyunsaturated fatty acid; PMS, Post mitochondrial supernatant; RBC, red blood cell; SFA, Saturated fatty acid; WAT, White adipose tissue.

E-mail address: vincent.rioux@institut-agro.fr (V. Rioux).

Keywords: Essential fatty acids; Dietary pentadecanoic acid; Odd chain fatty acids; Essential fatty acid deficiency; n-8 odd-chain PUFAs.

<footnote><sup>a</sup> Laboratory of Biochemistry and Human Nutrition, Institut Agro Rennes-Angers, Rennes, Bretagne, France</footnote>

<footnote><sup>b</sup> Institut Numecan, INRAE, INSERM, Univ Rennes, Rennes, Bretagne, France</footnote>

<footnote>* Corresponding author at: Vincent Rioux, Institut Agro Rennes-
Angers, 65 rue de Saint Brieuc, 35000 Rennes, Bretagne, France.</footnote>
a primer of fatty acid de novo synthesis in rumen bacteria [4,5]. Therefore, OCFAs are synthesized in rumen bacteria, absorbed in the intestine, then incorporated in milk fat in the mammary gland. Saturated OCFAs constitute minor SFAs in ruminant milk (1% of total milk fatty acids (FAs) for C15:0 and 0.5% for C17:0) [6]. They are often considered as specific of dairy, being described as plasma and adipose tissue biomarkers of dairy fat consumption [7,8]. The dietary intake of C15:0 appears to be dependent on the consumption of dairy fat, as being the major source of this fatty acid. Even though saturated OCFAs were discovered in the middle of the 20th century as part of a specific compound of dairy fat, their roles and physiological effects have begun to be studied only recently. During the 20th century, dairy fat and more specifically saturated dairy FAs have been considered detrimental to good health, e.g., with correlations between saturated fat and CVD risks. Yet, an increasing number of studies are now correlating that consumption of dairy product is associated to lower risks to develop pathologies, like type II diabetes, non-alcoholic fatty liver disease and CVD [9,10]. Furthermore, higher serum concentrations of C15:0 (as a marker of dairy fat intake) were found associated with lower risk to develop these pathologies, leading to associate this OCFA with beneficial nutritional effects [9,11–13]. A recent study showed C15:0, as a bacterial metabolite of inulin fermentation, is protective against non-alcoholic steatohepatitis (NASH) development in mice, through notably restoration of gut barrier function in diet-induced NASH [14]. Further, this study highlighted that pentadecanoic acid could be synthesized from the digestion of soluble fibers by the gut microbiota in mice, leading to new interrogations regarding endogenous pentadecanoic acid. Recently, beneficial effects of *in vivo* C15:0 supplementations were described both in high fat, high cholesterol diet fed rabbits and high fat diet fed mice, with an attenuation of the appearance of several pathologies like fibrosis, hypercholesterolemia and inflammation [15]. Henceforth, the literature is gaining arguments concerning the hypothesis that C15:0 might have protective effects against metabolic syndrome disorders. Furthermore, an endogenous C15:0 metabolite, pentadecanoylcarnitine, was newly suggested as an endocannabinoid with potential involvement in the regulation of inflammation, mood and sleep [16]. More, recently several studies led by Venn-Watson *et al*. have introduced the hypothesis that C15:0 may possess the suitable characteristics and metabolic functions to be considered essential for human nutrition [15,16].

As endogenous cellular synthesis of C15:0 is still unclear and may occur exceptionally in minor proportions through α-oxidation, through the presence of high levels of propionic acid [17] or through fiber digestion from the gut microbiota [14], dietary intake of dairy C15:0 seems to be the main way leading to its significantly increased levels in animal tissues, except in the lactating mammary gland of ruminants. Therefore, the hypothesis of essentiality of dietary pentadecanoic acid deserves further investigations in light of its physiological roles and absence of sufficient endogenous synthesis. That is why we aim to assess the effect of pentadecanoic acid supplementation on essential fatty acid deficiency. Historically, 3 FAs have been discovered as essential according to important physiological roles and lack or insufficient endogenous synthesis. Linoleic acid (C18:2 n-6, LA) and α-linolenic acid (C18:3 n-3, ALA) were the first described essential fatty acids (EFA) and later docosahexaenoic acid (C22:6 n-3, DHA) was also defined as essential, due to its very low synthesis starting from dietary ALA [18,19].

In this study, we aim to assess the claim of C15:0 potential essentiality using a rodent model of EFA deficiency, EFA deficient fed Wistar rats *in vivo*. Indeed, as there exists redundancy effects between EFAs (e.g., between LA and ALA), we novelly assessed C15:0 potential essentiality in absence of EFAs in the diet

to observe if C15:0 could induce compensatory effects to counteract some EFA induced symptoms. The model used to induce EFA deficiency was as follows: rat dams were fed an EFA-deficient diet 2 weeks prior to mating, then throughout gestation and until the neonates were weaned [20]. The impact of the deficiency was assessed in the pups at weaning. Then, pentadecanoic acid was supplemented in the diet (2% of dietary energy) to investigate changes in EFA-deficient symptoms mainly characterized by growth stunt, skin abnormalities and accumulation of a biochemical marker (Mead acid, C20:3 n-9) in tissues [21–23]. Physiological parameters were measured to assess the deficient state of pentadecanoic acid-supplemented animals and FAs analysis were conducted to determine pentadecanoic acid's metabolism under EFA deficiency.

## 2. Material and methods

### 2.1. Chemicals and fatty acids

All chemicals were purchased from Sigma Aldrich and solvent reagents were purchased from ThermoFisher Scientific. Commercial oils containing high levels of ALA (50 % in linseed oil) and LA (70% in sunflower oil) were used to produce ALA and LA ethyl esters. Linseed oil and sunflower oil were refluxed with a mix of ethanol-H₂SO₄ (10/1, V/V) for 2 h, under argon atmosphere, to convert triglycerides into fatty acid ethyl esters (FAEE). After hydrolysis, FAEE were extracted twice with hexane, washed, and dried with Na₂SO₄. The solvent was removed by evaporation under pressure. FAEE were separated by preparative chromatography as described elsewhere for other FAs [24]. Collected ethyl linoleate and linolenate were analyzed by gas chromatography-mass spectrometry (GC-MS) to check purity (>97%) then stored at -20°C under argon atmosphere to limit oxidation. Pentadecanoic acid was purchased from TCI Europe and converted into FAEE by the reactions described above.

### 2.2. Animals and diets

Animal procedures were performed according to the French and European directives and after validation by the ethics comity of Rennes and ministry of education (APAFIS #39726-2022112509248162 v8). To induce an efficient EFA deficiency, we performed an experiment in 2 parts. The first one was the induction of the EFA deficiency and the second corresponded to the compensation of the deficiency with supplementation of specific FAs. An experimental chart flow is presented in Figure 1.

For the first part, EFA deficiency was induced in female Wistar rats, 2 weeks before mating, then throughout the gestation and lactation periods. Female Wistar rats were purchased in Janvier Labs (Le-Genest-Saint-Isle, France). Thirteen female rats (8 weeks old) were separated into two groups, a control group (n=3 rats) and a deficient group (n=10 rats). Control female rats were fed a diet containing EFA in adequate proportion with 2% energy of LA and 0.5% energy of ALA, according to nutritional guidelines [25]. The control diet was also supplemented with 2% energy of C15:0 to attest its metabolism under non deficient conditions. Deficient female rats were given a diet containing no EFAs nor C15:0. We chose to induce a double deficiency of EFA (LA and ALA), as it has been described that some symptoms of LA deficiency can be compensated for by the presence of ALA [20]. After the gestation, pups stayed with their dams for 3 weeks, corresponding to the lactation period in which they were fed with the dams' milk. Litters were fitted to 11 pups per dam for a homogenization of the dams' lactation. After the weaning (pups age = 21 d), dams were separated from the pups for 2 h and an intraperitoneal injection of 2U of
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Experimental design of the induction of EFA deficiency in rats (A), followed by LA or C15:0 supplementation after the weaning (B). Female rats received two diets, CTRL diet (with 2% energy LA, 0.5% ALA and 2% C15:0) and deficient diet (with no EFA) 2 weeks before conception and throughout the pregnancy (3 weeks) and lactation period (3 weeks). After the weaning, the offspring were fed with 4 different diets, CTRL and deficient diets described above, and deficient diet supplemented either with C15:0 or LA.</figcaption></figure>

All diets were normocaloric (3.9 kcal/g), isolipidic (lipid=5% total mass) and indicated fatty acid % represent their energy intake level. Fatty acids other than supplemented ones (LA, ALA, C15:0) came from totally hydrogenated sunflower oil (only C16:0 and C18:0). Rats were fed *ad libitum*, weights and food consumption were monitored twice a week throughout the postweaning period.

oxytocin was performed to stimulate the lactation. Milk samples were collected under isoflurane anesthesia, then dams were euthanized by exsanguination for collection of organ samples. Right after, eight male pups per group were euthanized by exsanguination under isoflurane anesthesia for tissue sampling.

The second part of the experiment consisted in evaluating the evolution of EFA deficiency in male rats after weaning. At 21 d, deficient rats were homogeneously divided (according to weight) into three groups of eight animals fed with the following diets: a deficient diet similar to the dam's diet (deficient group); a 2% energy C15:0-supplemented diet (C15:0 group); a 2% energy LA-supplemented diet (LA group). Control rats (n=8) were fed with the control diet. We assessed the deficiency and the effect of C15:0 or LA supplementation on the postweaning period for 11 weeks. At the end of the experiment, rats were fasted overnight and euthanized by exsanguination under isoflurane anesthesia for tissue sample collection. All samples were immediately frozen in liquid nitrogen and stored at -80°C until analysis.

During the whole experiment, all animals were fed *ad libitum* and had free access to water. Animal weights and food consumption were measured throughout the entire postweaning period, twice a week. All diets were homemade, from a mix of a synthetic lipid-free flour prepared at the INRAE Unit "Sciences de l'animal et de l'aliment" (Jouy-en-Josas, France), which compounds are described in **Table 1** and a semi-purified lipid mix constituted of a lipid common base of totally hydrogenated sunflower oil, supplemented or not with individual specific FAEE. FA compositions of the diets are described in **Table 2**. All diets were normocaloric (3.9 kcal/g), containing 20.8% energy from proteins, 67.7% energy from carbohydrates and 11.5% energy from lipids (5% of total diet mass).

## 2.3. Plasma quantification of insulin growth factor-1

Plasma quantification of insulin growth factor-1 (IGF-1) was performed using Quantikine ELISA kit, Mouse / Rat IGF-1 immunoassay (Biotechne) according to the manufacturer recommendations. Adequate plasma dilutions were performed to obtain measurements values in the yield of the standard curve values. Measures of optical density were performed using the POLARstar Omega microplate reader (BMG Labtech) set at 450 nm.

<table><caption>**Table 1**

Composition of the experimental diets (in mass and energy intake).</caption><thead><tr><th>Compounds</th><th>Percentage (mass)</th><th>Percentage of energy</th></tr></thead><tbody><tr><td>Chlorhydrique casein</td><td>20.0</td><td>20.5</td></tr><tr><td>L-cystine</td><td>0.3</td><td>0.3</td></tr><tr><td>Corn starch</td><td>42.9</td><td>44.0</td></tr><tr><td>Maltodextrin</td><td>7.1</td><td>7.3</td></tr><tr><td>Caster sugar</td><td>16.0</td><td>16.4</td></tr><tr><td>Cellulose</td><td>4.0</td><td></td></tr><tr><td>Vitamins Premix AIN-93M</td><td>1.0</td><td></td></tr><tr><td>Choline bitartrate</td><td>0.2</td><td></td></tr><tr><td>Minerals Premix AIN-93M</td><td>3.5</td><td></td></tr><tr><td>Lipids</td><td>5.0</td><td>11.5</td></tr><tr><td>Sum</td><td>100.0</td><td>100.0</td></tr></tbody></table>

The flour composition was made specifically for this experiment. All nutrients are present in adequate proportions except for the lipid blends which were subsequently added. The composition was designed for the lipids to be added at 5% of total mass of the diet (45 kcal/kg of diet, accounting for 11.5 % of total energy intake).

## 2.4. Lipid extraction and gas chromatography coupled with mass spectrometry analysis

Lipid extractions and derivations into fatty acid methyl esters (FAMEs) from various tissues of each animal were performed as described elsewhere [20]. An internal standard (C13:0) was used to quantify total fatty acids in the samples. FAMEs were analyzed by GCMS in electronic impact mode (GCMS EI). GCMS analysis was performed using an Agilent 7890A with a bonded silica capillary column (BPX 70, 60 m × 0.25 mm). Helium was used as carrier gas. The column temperature program started at 150°C and gradually increased at 4°C/min to 250°C and held at 250°C for 10 min. Mass spectra were recorded with an Agilent Technologies 5975C inert MSD with triple-axis detector. The mass spectrometer was operated under electron impact ionization conditions (electron energy 70 eV, source temperature 230°C). Data were obtained in full-scan mode with a mass range of m/z 50–550 amu. Identifica-
<caption>Table 2</caption>

FA composition (% of total FAs) of the experimental diets. CTRL: control diet containing essential FAs (LA and ALA) and C15:0; Deficient: diet deficient in essential FAs; C15:0: deficient diet supplemented only with C15:0; LA: deficient diet supplemented only with LA.

<table><thead><tr><th>FA composition</th><th>Control</th><th>Deficient</th><th>C15:0</th><th>LA</th></tr></thead><tbody><tr><td>Palmitic acid (C16:0)</td><td>9.1</td><td>15</td><td>12.4</td><td>12.4</td></tr><tr><td>Stearic acid (C18:0)</td><td>51.7</td><td>85</td><td>70.2</td><td>70.2</td></tr><tr><td>Linoleic acid (C18:2 n-6, LA)</td><td>17.4</td><td>0</td><td>0</td><td>17.4</td></tr><tr><td>Alpha linolenic acid (C18:3 n-3, ALA)</td><td>4.4</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Pentadecanoic acid (C15:0)</td><td>17.4</td><td>0</td><td>17.4</td><td>0</td></tr><tr><td>Sum</td><td>100.0</td><td>100.0</td><td>100.0</td><td>100.0</td></tr></tbody></table>

All diets have the same lipid base, composed with totally hydrogenated sunflower oil containing only saturated FAs (palmitic and stearic acids). Essential FAs are supplemented as fatty acid ethyl ester, in proportions described in nutritional guidelines (2% energy for LA and 0.5% energy for ALA). C15:0 is also supplemented as fatty acid ethyl ester in the same amounts as linoleic acid (2% of energy).

tions of FAMEs were performed according to the retention time of methyl ester standards when available and mass spectra data collected. FAMEs were analyzed using MassHunter Workstation Software Qualitative Analysis (version B.07.00 for Windows, Agilent Technologies).

## 2.5. 4,4 Dimethyloxazoline derivatives and analysis for identification of double bond positions

For the specific double bond position identification on unknown fatty acids, FAMEs were converted into 4,4 Dimethyloxazoline (DMOX) derivatives as previously described [26]. DMOX were analyzed by GC-MS EI with the same parameters described above.

## 2.6. Lipid species separation by thin layer chromatography

To separate lipid species, total liver lipids were extracted, evaporated and solubilized in chloroform. Thin silica plates (0.5 mm thickness) were homemade using 60H silica gel. Total lipids were added on the silica plate and migrated in a solvent saturated tank (hexane 85%, ether 14% and acetic acid 1%) for 45 min at room temperature. Lipid spots were revealed using primulin dye under UV then were extracted from the silica, saponified and methylated into FAMEs [27]. FAMEs from each lipid species were extracted twice with pentane, washed, evaporated and analyzed by GC-MS EI.

## 2.7. Desaturases activity measurements

In vitro enzymatic activity measurements of the Δ9-, Δ6- and Δ5-desaturases were performed [28,29]. Frozen livers (collected during euthanasia) were weighted, mixed using Potter homogenization in sucrose buffer (0.25 M in 50 mM PO₄ buffer, pH 7.4) and centrifuged at 10 000g for 15 min at 4°C. Postmitochondrial supernatants (PMS) were retrieved and incubated with FA substrates (60 µM) in the presence of desaturase reaction cofactors: magnesium chloride (MgCl₂, 0.15M), NADH (0.02 M), ATP (0.09 M) and CoA sodium salt (0.014 M). Four deuterium (²D) stable isotope FAs were used to measure specific product apparition [30]. For Δ6-desaturase activity assessment, ²D5-C18:2 n-6 and ²D5-C18:3 n-3 were used. For Δ5-desaturase activity assessment, ²D6-C20:3 n-6 and ²D8-C20:4 n-3 were used. For the Δ9-desaturase activity measurements, three saturated substrates were used (C15:0, C16:0 and C17:0) to compare the activity among substrates. Reaction mix were incubated 20 min at 37°C and reactions were stopped by addition of 1 mL of 12% KOH. To assess reaction quality, a control assay was also run by stopping the reaction before addition of the substrate. Total lipids were saponified, extracted and washed. Solvents were evaporated and pentafluorobenzyle (PFB) ester derivation was achieved by addition of PFB solution (7% in acetonitrile)

and triethylamine (TEA) with a 10:1 ratio [31]. Reaction was completed at room temperature for 15 min, reagents were thoroughly evaporated under nitrogen flux, for 20 min. Finally, PFB esters were analyzed by negative chemical ionization (NCI) GCMS on a 7890 A GC system coupled to a 5977B MS detector [32]. PFB esters were separated using a bonded silica capillary column (BXP70, 60m x 0.25mm) and helium as carrier gas. The column temperature (started at 190°C) was gradually increased by 4°C/min to 250°C and held for 10 min. The mass spectrometer was operated in NCI mode using methane as the ionization gas. Acquisitions were performed using selected ion monitoring (SIM) mode. FAs were identified according to the molecular ion, retention time and PFB ester standards. PFB esters were analyzed using MassHunter Workstation Software Qualitative Analysis (version B.07.00 for Windows, Agilent Technologies). FA products quantified in the control assay were subtracted from the ones quantified in measurement assays.

## 2.8. Statistical analysis

All statistics were made using Graph 8 Prism. Either t-test or ANOVA with multiple comparisons were made when assumptions of normal distribution and homogeneity of variance were verified using respectively Shapiro-wilk and Brown-Forsythe tests. If not, Mann-Whitney or Kruskal-Wallis tests were performed.

For growth rate statistical comparisons, linear regressions were performed, goodness of fit was assessed with a R square between 0.94 and 0.96 for all groups. Best-fit regression models were compared using an ANOVA on slope values and SD.

# 3. Results

## 3.1. The induction of EFA deficiency in the dams leads to strong deficiency markers in the weaned pups

For the first part of the experiment (Fig. 1), the aim was to obtain EFA-deficient animals by inducing an EFA deficiency in female rats through the diet and obtaining an EFA-deficient offspring at weaning. After 8 weeks of diet, dams' weights were not significantly different between control and deficient female rats (Fig. 2A). FA composition analyses of liver and red blood cells (RBC) were made to determine the presence of specific FAs used as markers of EFA-deficiency. Mead acid (MA) and arachidonic acid (ARA) values were analyzed and the ratio MA / ARA was calculated (Fig. 2B in RBC and C in liver). In RBC, deficient dams' MA / ARA ratios were significantly higher compared to CTRL dams. The same observation was seen in the liver. EFA deficiency was also characterized in the dams' milk by important decreased amount of EFA in deficient group compared to control (Fig. 2D). Deficient milk displayed a 84% decrease of LA amounts and a 97% decrease of ALA amounts.
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

B

<figure><img src="image_6.png" /></figure>

RBC

C

<figure><img src="image_7.png" /></figure>

Liver

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

Milk

**Figure 2.** Characterization of EFA deficiency markers in dams after 8 weeks of diet. (A) Weights of dams at the end of the lactation period (age 16 weeks); (B) Mead Acid /Arachidonic acid ratio measured in RBC; (C) Mead Acid /Arachidonic acid ratio measured in liver; (D) FA composition of milk collected just before euthanasia; (E) Total mass of FAs in milk.
Values were obtained from relative quantification of FAMEs in total lipid tissues by GCMS EI. Data are represented as mean ± SEM. Statistical significances between two groups are represented with different letters.

Pentadecanoic acid and other OCFAs were also detected in control milk and only traces or absence in deficient milk. Finally, the milk total FA mass did not significantly differ between control and deficient group (Fig. 2E).

EFA deficiency induction in dams did not alter reproduction nor gestation and no mortality was observed in dams nor pups. The size of the litters was not significantly impacted by the diet (supplementary Fig. 1). At 3 weeks of age, male pups born from EFA-deficient dams, i.e., EFA-deficient pups displayed a body weight 23% lower than that of pups born from CTRL dams, i.e., CTRL pups (Fig. 3A). Quantification of plasma insulin growth factor 1 (IGF-1) was performed to further assess the impairment of growth in the pups (Fig. 3B). As expected, deficient pups displayed a lower IGF-1 plasma concentration than control pups. Similar to deficient dams, deficient pups had higher MA / ARA ratios than control pups after both RBC and liver FA analysis (Fig. 3C and D). Interestingly, RBC ratio value of deficient animals was similar in dams and in pups

whereas in the liver, the value of the ratio seemed to be higher in deficient pups than in deficient dams.

To conclude on this first part, the 2 parameters analyzed here, growth and biochemical marker of deficiency, revealed deficient group pups presented strong EFA deficiency markers. Therefore, we validated the induction model of EFA deficiency in weaned pups.

## 3.2. C15:0 dietary supplementation in EFA-deficient animals increases body growth rate during 5 weeks after weaning

For the second part of the experiment (Fig. 1), i.e., postweaning period, rats were fed either control, deficient or specific FA-supplemented (C15:0 or LA) diets for 11 weeks. Food consumptions were measured throughout the entire period, twice a week, to ensure animals consumed the same amount of food and therefore lipids and calories (supplementary Fig. 2). Weights were measured twice a week until the end of the experiment (Fig. 4A). Analysis of
<figure><img src="image_11.png" /><figcaption>**Figure 3.** Characterization of EFA deficiency markers in pups at weaning. (A) Weights of pups (age 3 weeks); (B) IGF-1 plasma concentration; (C) Mead Acid /Arachidonic acid ratio measured in RBC; (D) Mead Acid /Arachidonic acid ratio measured in the liver. Values were obtained from relative quantification of FAMEs in total lipid tissues by GCMS EI. Plasma concentration of IGF-1 was determined using Immunoassay ELISA kit on plasma collected at euthanasia. Data are represented as mean ± SEM. Statistical significances between two groups are represented with different letters.</figcaption></figure>

the growth was first performed during the early postweaning period (d 3-34) where the growth rate is considered constant and at its highest. Linear regressions were performed (Fig. 4A) to evaluate the growth rate values (Fig. 4B). As expected, EFA-deficient rats displayed a significantly lower growth rate than control group. LA-supplemented rats had a higher growth rate than deficient. Interestingly, C15:0-supplemented rats had a similar growth rate than LA, that was significantly increased compared to deficient rats. Yet, C15:0 or LA supplemented rats had significantly lower values than control group. At 11 weeks postweaning (end of the second part of the experimental design), EFA-deficient rats still displayed a significant 17% lower body weight as compared to control rats (Fig. 4C). C15:0 supplemented rats' weight was increased, although not significantly, compared to deficient ones. Interestingly, the same effect was observed with LA supplemented rats with no significant effect on the body weight compared to deficient group. Nonetheless, C15:0 and LA supplemented rat body weights were also not significantly different to control. IGF-1 plasma concentrations were also determined at the end of the postweaning period (Fig. 4D). As observed in weaned pups, deficient rats still displayed a lower IGF-1 plasma concentration than control group. C15:0 and LA supplementation also displayed lower concentrations than control group, and similar values as deficient.

### 3.3. Under EFA deficiency characterized with altered FA metabolism, postweaning C15:0 supplementation induces the synthesis of the new PUFA family of n-8 OCFAs

At 11 weeks postweaning, rats were euthanized for organ and tissue collection. MA / ARA ratio analyses were performed on RBC and liver by total lipid extractions and FA composition analyses (Fig. 4E and F). As observed at weaning, EFA-deficient rats still displayed a significant increase in the ratio compared to control ones, in both RBC and liver. LA supplemented rats had a significantly lower ratio than deficient group, with mean values equivalent to control group. As expected, the ratio wasn't decreased for C15:0 supplemented group, with no significant difference compared to deficient rats.

OCFAs compositions were analyzed in liver to investigate C15:0 metabolism under different EFA nutritional status. Indeed, as expected OCFAs were detected as traces in groups for which the diet did not contain C15:0. Here, we compared OCFAs between CTRL and C15:0 supplemented group, i.e., the two diets containing C15:0 (Fig. 5A). First, a significant 40% decrease of the presence of C15:0 in C15:0-supplemented rats' liver was surprisingly observed, compared to CTRL rats. As a result, a differential metabolic conversion of C15:0 was observed with a significant less amount of C17:0
<figure><img src="image_12.png" /><figcaption>**Figure 4.** Effect of the FA supplementations on pups during the postweaning period: growth and Mead acid / Arachidonic acid ratio. (A) Body weights of the rats during the whole postweaning period (11 weeks starting from age 3 d); (B) Growth rate of the rats during the beginning of the postweaning period (3-34 d); (C) Weights of the rats at the end of the experiment (80 d); (D) IGF-1 plasma concentration at the end of the experiment (80 d); (E) Mead Acid /Arachidonic acid ratio measured in RBC; (F) Mead Acid /Arachidonic acid ratio measured in the liver.</figcaption></figure>

Weights were measured twice a week during the postweaning period. Plasma concentration of IGF-1 was determined using Immunoassay ELISA kit on plasma collected at euthanasia. Multiple linear regressions were performed on the weights of the rats between 3 and 34 d and regression slopes were compared to observe differences of growth rate. Ratio values were obtained from relative quantification of FAMEs in total lipid tissues by GCMS EI. Data are represented as mean ± SEM. Statistical significances between two groups are represented with different letters.
<figure><img src="image_13.png" /><figcaption>**Figure 5.** Quantification and identification of odd-chain fatty acids in the liver. (A) Quantification of odd-chain saturated, monounsaturated and polyunsaturated fatty acids in the liver of rats fed the CTRL and C15:0 diets; (B) Mass spectrum of the DMOX derivative of the unknown C19:3, leading to its identification as C19:3 n-8; (C) Mass spectrum of the DMOX derivative of the unknown C21:3, leading to its identification as C21:3 n-8; (D) Mass spectrum of the DMOX derivative of the unknown C21:4, leading to its identification as C21:4 n-8.</figcaption></figure>

Results are expressed as % of total FAs. Only the two rat groups containing C15:0 in their diets (CTRL and C15:0 diets) are shown. Total FAMES of liver C15:0 group rats were converted to 4,4 Dimethyloxazolyne derivatives (DMOX) to specifically analyze double bond positions. DMOX were analyzed by GCMS EI. Values were obtained from relative quantification of FAMES in liver total lipids by GCMS EI. Data are represented as mean ± SEM. Statistical significances between two groups are represented with different letters.

but higher amount of C17:1 n-6 in C15:0 group. A significant less amount of C17:1 n-8 was also observed in C15:0 group compared to CTRL. Only in C15:0 group, the presence of odd chain PUFAs (OCPUFAs) was detected, notably an unknown C19:3 and nonquantifiable traces of C21:3 and C21:4. The structural characterization of these three OCPUFAs was performed using DMOX derivatives for specific determination of double bond positions. Δ5, Δ8, Δ11-C19:3 n-8, Δ7, Δ10, Δ13-C21:3 n-8 and Δ4, Δ7, Δ10, Δ13-C21:4 n-8 were specifically identified in the liver of C15:0 rats (Fig. 5B-D). Further investigations were conducted to identify in which lipid

species these odd chain n-8 PUFAs were esterified. After thin layer chromatography separation of C15:0 rats' liver lipid species, C19:3 and C21:3 n-8 were detected only in phospholipids (supplementary Fig. 3).

Concerning the other PUFA families (classical ones apart from the new described n-8), FA analyses were performed on specific tissues to complete the assessment of the impact of EFA deficiency and specific FA supplementation (Fig. 6). Six tissues were targeted according to their known FA composition specificity. The liver was analyzed as the organ responsible for important FA metabolism, *de*
<figure><img src="image_14.png" /><figcaption>**Figure 6.** FA tissue compositions of the rats fed the four experimental diets, regarding the presence or not of EFAs. Results are expressed as % of total FAs for each conventional family of PUFAs (n-3, n-6, n-7 and n-9). The following tissues were analyzed: (A) Liver; (B) Testis; (C) Brain; (D) Retina; (E) Tongue; (F) WAT. Fatty acid percentages were obtained from relative quantification of FAMEs by GC-MS EI. Data are represented as mean ± SEM. Statistical significances between two groups are represented with different letters.</figcaption></figure>

novo synthesis and regulations. The testis was targeted as a n-6 rich organ, brain and retina as n-3 rich organs and the tongue as n-3 and PUFAs rich organ. White adipose tissue (WAT) was analyzed for the storage forms of lipids. In all tissues analyzed, EFA-deficient, C15:0 and LA-supplemented animals displayed a strong decrease of n-3 PUFAs, consistent with ALA being absent in the diets. In brain, retina and tongue (Fig. 6C-E), the deficiency was even more marked with drastic diminution of n-3, e.g., the tongue of deficient animals, which displayed a 75% decrease of total n-3 compared to control group. The effect of the EFA-deficiency on n-6 PUFA content was differential depending on the n-6 specificity of the tissue. In the liver, testis, tongue and WAT, deficient and C15:0 rats had a lower proportion of n-6 PUFAs than control rats (Fig. 6A-F). Whereas interestingly, in brain and retina, n-

6 proportions were higher in deficient and C15:0 animals than in control animals. For LA supplemented animals, rats displayed similar n-6 proportions in testis and WAT than control animals. Noteworthy, the proportions were higher in LA supplemented animals in the liver, brain, retina and tongue compared to control rats. EFA-deficiency and C15:0-supplementation significantly increased n-7 and n-9 PUFAs proportions as compared to control rats in all tissues except brain whereas LA-supplementation had no effect as compared to control animals, on n-7 and n-9 PUFAs.

In order to better understand the liver PUFA profiles shown in Fig. 5 and Fig. 6A, measurements of liver Δ6- and Δ5-desaturase activities were performed (Fig. 7). EFA-deficient and C15:0-supplemented rats had lower liver activities of both enzymes than other groups, but still retained the metabolic capacity
<figure><img src="image_15.png" /><figcaption>**Figure 7.** Effect of the four experimental diets containing or not EFAs on liver desaturase activities.</figcaption></figure>

(A) Δ6-desaturase activity assessment using LA as substrate. (B) Δ6-desaturase activity assessment using ALA as substrate. (C) Δ5-desaturase activity assessment using C20:3 n-6 as substrate. (D) Δ5-desaturase activity assessment using C20:4 n-3 as substrate. Activities were determined *in vitro* from sample of frozen adult rat liver. The 2 main known substrates of each desaturase were used to assess the activities, using stable isotope FAs as substrates. Analyses of reaction product synthesis were performed by GCMS-NCI. Data are represented as mean ± SEM. Statistical significances between two groups are represented with different letters.

to introduce double bonds on known substrates of Δ6- and Δ5-desaturases. LA-supplemented rats had restored activities levels of both desaturases similar to that of the control rats.

To conclude, C15:0 supplementation at weaning induced the synthesis of n-8 OCPUFAs, with a newly characterized metabolic suspected pathway.

## 4. Discussion

In this study, we aimed to assess the potential effects of pentadecanoic acid to restore at least partly EFA deficiency symptoms. We showed that C15:0 supplementation in EFA deficient rats increased early growth rate and lead to the synthesis of original n-8 OCPUFAs.

EFA deficiency has been characterized since Burr and Burr pioneer work on fat-deprived rats [21]. Early studies showed EFA-deficient animals displayed characteristic morphological and biochemical markers. In our work, EFA deficiency symptoms observed in rats were similar to the ones described in the literature [20,21]. As expected, no significant differences were observed on the weight of dams after 8 weeks fed on EFA-deficient diet compared to control diet fed dams (Fig. 2), since adult rats have lower growth rate than young individuals. However, there was a weight difference at weaning between CTRL and EFA-deficient fed pups (Fig. 3). Indeed, EFA-deficient pups had developed a strong stunt growth symptom. In accordance to weight observation, IGF-1 plasma concentrations were reduced in EFA-deficient animals compared to control ones.

Here, growth was assessed throughout 2 factors: weight measurements and IGF-1 plasma levels. IGF-1 is a liver-synthesized growth factor involved in bone and muscle growth [33]. Its measurement has been described as a reliable way to evaluate the growth hormone (GH) levels [34]. Indeed, as opposed to the GH synthesis which is pulsatile, IGF-1 synthesis is constant throughout the day and plasma levels are a great reflection of the GH secretion by the pituitary gland. Hence, IGF-1 is considered as a growth hormone in mammals, and its diminution or absence has been shown to cause growth retardation in mice [34]. Noteworthy, other factors would have been interesting to measure for further assessment of the growth in animals, e.g., IGF binding protein, vascular endothelial growth factor or transforming growth factor. These results enhance the fact that to obtain EFA-deficient animals with growth stunt symptoms, it is necessary to work with young individuals obtained from EFA-deficient diet fed mothers before and during gestation and lactation. Noteworthy, here the assessment of the EFA deficiency within each time period (from preconception to weaning) was not performed yet it would be interesting in future research to determine which period is impacting the most such phenotypes. Likewise, the weight of the pups at birth was not measured (to not interfere with the bound between dams and pups, to avoid cannibalism or abandonment of the pups). It would be interesting to determine if the EFA deficiency during pregnancy impacted the weight of the pups at birth or if the lactation period had a higher impact.

The stunt growth observed at weaning was conserved at the young adult stage (14 weeks old). While the absolute weight difference between CTRL rats and EFA-deficient rats increased
between weaned rats and adult rats (P=.001, weaned=18.3±5.7 g; adults=80.0±38.8 g), the relative percentage of difference between the weights did not (P=.10, weaned=23.34±7.20%; adults=16.67±8.08%). Therefore, the stunt growth observed at the weaning in EFA-deficient rats did not increase during the post-weaning period (Fig. 4). Since the supplementation of C15:0 or LA started after the weaning, the limit of this model was the short period of action of FAs of interest to restore growth in animals. Indeed, there was a decrease of the growth rate in all groups around 35 d postweaning, inducing a limited time of action of the supplementation of C15:0 or LA. This may explain why the supplemented animals did not have a different weight 11 weeks postweaning as compared to EFA-deficient animals. Yet, despite their limited effects after the weaning, when comparing growth rates, C15:0 supplemented diet did induce an increased growth in animals compared to EFA-deficient 1 (Fig. 4B), and this effect was found similar in LA-supplemented animals. Indeed, in our in vivo model, C15:0-supplementation had a positive effect on the growth in response to EFA deficiency in the rats. This effect on growth was observed during the juvenile period in rats, between weaning and sexual maturity (21 and 50 d of age). This period in human life would correspond to the following time period: 6 months to 11.5 years of age [35]. The beginning of the juvenile period is considered critical (1,000 first d in humans) as it represents the major period of development in which adequate nutritional intakes are vital to enhance children's growth and health [36]. Since C15:0 can positively affect the growth during this early period of life and because it is a specific FA of milk and dairy products, C15:0 could be a direct or indirect (through its metabolites) actor in early development.

The second parameter for the evaluation of the EFA deficiency was the value of the MA / ARA (Mead acid, C20:3 n-9 and Arachidonic acid, C20:4 n-6) ratio in the RBC and in the liver. EFA-deficient (more specifically LA deficient) animals synthesize lower amounts of ARA and more generally lower amounts of omega 6 PUFAs derivatives. To compensate for a lower amount of PUFAs (needed for physiological functions but also for structural ones), the body induces a high synthesis of MA through the n-9 family, from oleic acid. In this case, a lower amount of ARA and higher of MA results in a higher MA / ARA ratio in RBC and also in liver [37]. In our study, in both weaned and adult stages (Fig. 3C and D and Fig. 4E, F), EFA-deficient animals displayed an important increase in the ratio compared to control group, in RBC and liver total lipids. Noteworthy, this ratio in pups at weaning and dams, in the liver, was different. This is most likely due to differences in the physiological state, the requirement of PUFA synthesis (in response to a higher phospholipid membrane synthesis during growth) and the reservoir of EFA in animals. Usually, the MA / ARA is defined in RBC, here we showed that RBC and liver exhibited the same ratio trends in all groups. However, ratios seemed to be higher in the liver. As liver is the main organ for FA synthesis, the elevated values of the ratio compared to the one in RBC are consistent. After weaning, LA-supplementation restored the MA / ARA ratio to that of control animals (Fig. 4E and F). Indeed, in LA-supplemented rats, the presence of the n-6 precursor consistently resulted in higher ARA amounts and lower MA amounts in the liver compared to EFA-deficient animals. Therefore, in addition to the stunt growth syndrome, EFA-deficient animals displayed a strong marker of EFA deficiency. The supplementation of LA completely restored the MA / ARA yet C15:0 did not and wasn't different from EFA-deficient group on this parameter.

To assess the EFA deficiency beyond the MA / ARA ratio, FA compositions were analyzed on EFA-rich organs (Fig. 6). Analyzes confirmed that EFA-deficient fed rats developed significant re-

ductions in n-3 and n-6 FAs amounts. WAT composition analyses showed n-3 and n-6 FAs stocks were highly reduced after being fed EFA-deficient diet for several weeks. Concomitant to drastic reductions of EFA storage, EFA-rich organs displayed strong depletions of EFA amounts. Associated with EFA reductions, in our study EFA-deficient rats displayed lower Δ5 and Δ6 desaturase liver activities (Fig. 7). LA and ALA enrichment in the diet has been shown to increase Δ5 and Δ6 desaturases activities [29]. In our model, lower proportions of EFA could explain lower desaturases activities. Besides, to corroborate LA-supplementation resulted in an increase of the desaturase activities compared to EFA-deficient group. However, other studies reported that EFA-deficient diet increased their activities in rats [38,39]. Regulations of the Δ5 and Δ6 desaturase activities by dietary PUFAs appear to be controversial in the literature and activity measurements results seem highly dependent on the diet composition and the control diet used as a comparison, as well (fat free, amounts of lipids) [40]. Noteworthy, the assessment of elongases activities was not performed here as only delta 5 and 6 desaturase activities were. Indeed, delta 5 and more specifically delta 6 desaturase are responsible for rate-limiting steps in the conversion pathway of PUFA (like LA) into long chain PUFA or highly unsaturated fatty acids as opposed to elongation steps often yielded by a redundancy of elongases enzymes. Likewise, Δ9 desaturase activity could not been assessed as because animals were fasted overnight to euthanasia, which highly reduces the activity of delta 9 desaturase in the liver [41]. Nonetheless, an assessment of elongases impact would be interesting to further characterize this model.

As a response to the reduction of n-3 and n-6 PUFAs, n-7 and n-9 PUFAs were elevated in EFA-deficient animals (Fig. 6). This is consistent with the increase in MA observed in our study and in the literature [21–23]. N-7 and 9 FAs were consistently the only PUFAs which could be synthesized *de novo* or from the only 2 FAs present in the diet (palmitic and stearic acids) to compensate the lack of n-3 and n-6 PUFAs.

The supplementation of C15:0 in EFA deficient rats had no effect on the EFA deficiency biochemical parameters described above. As we expected, C15:0 did not change the MA / ARA ratio nor the proportions of EFA and desaturase liver activities. These alterations were caused by a specific lack of n-6 and n-3 PUFAs. The lack of PUFA is most likely responsible for structural needs regarding the MA/ARA ratio rise. While the lack of conversion and regulation of the delta 5 and 6 desaturase of the essential PUFA (e.g., LA) into long chain PUFA (e.g., ARA) would be responsible for the lower activities of the delta 5 and 6 desaturases.

While EFA-deficient rats appeared to compensate the unavailability of n-6 and n-3 by the synthesis of n-7 and n-9 FAs, C15:0 supplementation seemed to provide an increased diversity of PUFAs with its conversion into OCFA metabolites, especially n-8 FAs. Nonetheless, this conversion doesn't appear sufficient to counteract the elevation of MA/ARA ratio nor to modulate the activity of the delta 5 and 6 desaturases. Indeed, as a saturated fatty acid, the conversion of C15:0 into PUFAs is most likely less efficient than the conversion of LA into ARA. Thus, this could be a hypothesis explaining why C15:0 did not decrease the MA / ARA ratio as the body still needs PUFAs for membranes. Concerning the delta 5 and 6 desaturases, an explanation could be that C15:0 does not modulate at all their activity, as LA and its conversion into ARA does.

Nonetheless, C15:0 has been converted into several OCFA metabolites (Fig. 5). Mainly C17:0, n-6 and n-8 OCFAs were found synthetized from dietary C15:0. Yet, only n-8 and not n-6 odd-chain PUFAs, were identified (Fig. 5B-D) and incorporated into membrane phospholipids. To our knowledge, this is the first time these fatty acids have been characterized *in vivo*. Therefore, we here propose a suspected pathway of odd-chain n-8 PUFA synthe-
<figure><img src="image_16.png" /><figcaption>**Figure 8.** Proposed suspected pathway of odd-chain n-8 PUFA synthesis, starting from dietary C15:0 in EFA deficient rats. Starting from C15:0 supplemented in the diet, black colored FAs represent fully-characterized FAs. Grey colored FAs represent potential intermediary FAs, probably not detected because of their very low level and rapid conversion in this biosynthesis suspected pathway. $\varepsilon$ represents elongation step, $\Delta$ above arrows represent desaturation step, $\Delta$ under FAs represent double bond positions.</figcaption></figure>

sis, starting from dietary C15:0 in EFA deficient rats (Fig. 8). The $\Delta$ position of double bounds of detected FAs (determined by DMOX derivatives analyses) was used to hypothesize the structure of potential intermediary FAs, probably not detected because of their very low amounts and rapid conversion in this biosynthesis suspected pathway. This suspected pathway starts by the elongation of C15:0 into C17:0, then C17:0 is desaturated by the $\Delta9$ desaturase into C17:1 n-8, the first FA of the n-8 family. The next detected FA is the first odd chain n-8 trienoic fatty acid, $\Delta5$, $\Delta8$, $\Delta11$-C19:3 n-8. The suspected conversion steps from C17:1 to C19:3 are described as follow: $\Delta6$ desaturation, elongation, $\Delta5$ desaturation. Noteworthy, these suspected conversion steps mimic the ones described for the conversion of LA to ARA or ALA to EPA. At last, two odd chain n-8 tetraenoic fatty acids, $\Delta7$, $\Delta10$, $\Delta13$-C21:3 n-8 and $\Delta4$, $\Delta7$, $\Delta10$, $\Delta13$-C21:4 n-8 represented the longest n-8 OCFAs. These very specific FAs are likely to have been synthesized by 2 successive elongations from C19:3, followed by $\Delta6$ desaturation and partial $\beta$-oxidation. Once more, this suspected pathway is notably the same described for the conversion of ARA into C22:5 n-6 or EPA into DHA. Thus, as stated this is a suspected pathway, future investigations are needed to fully characterize it notably through the use of inhibitors or knockouts of the desaturases combined with the use of chemically synthesized n-8 odd chain PUFAs, as they have been novelly discovered and aren't commercially available.

In our model, only n-8 OCFAs were found multiple times desaturated into PUFAs as opposed to n-6 OCFAs which were not. Contrary to expectations, n-6 OCFAs did not appear responsible for essential functions as belonging to the n-6 family could had been suggesting. The preferred n-8 pathway could be explained by different enzymatic limiting rate steps in the OCFAs metabolism. Indeed, C15:0 was found to be elongated into C17:0 (Fig. 6), appearing as the first main product of C15:0 metabolism. The conversion of these two saturated OCFA into n-6 or n-8 unsaturated OCFA is thereafter performed by the $\Delta9$-desaturase enzyme. Therefore, we additionally determined the activity of the $\Delta9$-desaturase in presence of C15:0 and C17:0 substrates by measuring desaturation products (Fig. 9). Noteworthy, in our study, adult rats were food-deprived overnight before euthanasia, which highly reduces $\Delta9$ desaturase activity [42,43]. Hence, to avoid biased measurement of this activity, we determined it using liver from fed control pups obtained at weaning.

C15:0 was less desaturated into C15:1 n-6 than C17:0 was into C17:1 n-8, visualized by a higher activity (and higher affinity) of the $\Delta9$-desaturase for C17:0 than C15:0, like it has also been described for even-chain SFAs, since stearic acid is the preferred substrate compared to palmitic acid [44]. These results could explain the fact that n-8 OCFAs are further synthesized than n-6 OCFAs, due to the competition for the first step of $\Delta9$-desaturation. Noteworthy, lauric acid has been previously found to be a precursor of n-3 FAs by $\Delta9$ desaturation into C12:1 n-3 [44]. Thus, under specific conditions, synthesis of n-3 or n-6 FAs can be performed in mammals using saturated substrates like lauric acid (into C12:1 n-

<figure><img src="image_17.png" /><figcaption>**Figure 9.** $\Delta9$-desaturase activity increases with substrates carbon length among odd chain saturated fatty acids. $\Delta9$ desaturase activities were determined *in vitro* using frozen control rat liver collected at weaning and incubated with different saturated FA substrates (C15:0, C16:0 and C17:0). Analyses of reaction product synthesis (C15:1 n-6, C16:1 n-7 and C17:1 n-8) were performed by GCMS-NCI. Data are represented as mean ± SEM. Statistical significances between two groups are represented with different letters.</figcaption></figure>

3) and pentadecanoic acid (into C15:1 n-6). Noteworthy, the affinity of the delta 5 and 6 desaturases for C15:0 was not assessed here. One study described that C15:0 was not a substrate of the delta 5 and 6 desaturases, yet no desaturase activities assessments were performed [45]. Further investigations are needed to conclude on the affinity of delta 5 and 6 desaturases, and their potential isoforms [46], for C15:0, notably throughout the assessment of desaturase activities measurements.

OCPUFAs of n-8 family could be interesting actors in physiological functions and could be responsible for the increased growth observed in C15:0 rats. Indeed, C19:3 n-8 has an interesting double bond in $\Delta8$ position, which is required for some oxylipin synthesis [47,48]. Therefore, C19:3 n-8 synthesized from C15:0 could be a precursor of oxygenated mediators, as "nonadecanoids" (19 carbon length eicosanoid analogues). Eicosanoids are known to play important roles in regulations of physiological functions, like growth for instance, with prostacyclin I2 which promotes proliferation and differentiation of adipose precursor cells [49]. Thus, potential nonadecanoids derived from C19:3 n-8 could be actors in the physiological effect observed.

This study has potential limitations. The effects estimated in this model are to be considered relative to the specific and extreme EFA deficiency induced here. Hence, the phenotypes observed in response of EFA deficiency (e.g., growth retardation) can be specific to the design of the study, for instance the time of deficiency (i.e., EFA deficiency from preconception to the end of lactation) and the moment of supplementation of either EFA or C15:0 and should be considered in consequence. Nonetheless, within the conditions of our study, the use of adequate control groups (non-EFA deficient group for the first part of experiment and implementation of a second control group supplemented in LA in the second
part) strengthen the validity of the phenotypes we described. Finally, due to the absence of commercial availability of purified n-8 odd chain PUFAs, the study of these newly discovered fatty acids cannot be performed currently, and their potential physiological effects remain hypothetical for now. Nonetheless, for future research needs, we aim to elaborate the chemical synthesis of these purified fatty acids to assess the effects of these odd chain PUFAs in future studies.

In summary, this study shows that pentadecanoic acid, a saturated OCFA, can restore some physiological parameters, such as growth, induced by EFA-deficient diet in the rat. The synthesis of a new family of FAs, n-8 OCPUFAs derived from dietary C15:0, is promising in the discovery of physiological effects of the OCFA family. Chemical synthesis of these PUFAs and further investigations are needed to better understand their action mechanisms and to decipher their place in human nutrition.

## Data availability statement

Data supporting the conclusion of this article are to be shared upon request.

## Declaration of competing interest

Vincent Ciesielski's PhD thesis is funded by the Centre National Interprofessionel de l'Economie Laitière (CNIEL, France). The other authors declare that there are no conflict of interest.

## CRediT authorship contribution statement

**Vincent Ciesielski:** Writing – original draft, Investigation, Formal analysis, Conceptualization. **Thomas Guerbette:** Writing – review & editing, Investigation. **Léa Fret:** Writing – review & editing, Investigation. **Mélodie Succar:** Writing – review & editing, Investigation. **Youenn Launay:** Writing – review & editing, Investigation. **Patrice Dahirel:** Writing – review & editing, Investigation. **Philippe Legrand:** Writing – review & editing, Investigation. **Manuel Vlach:** Writing – review & editing, Investigation. **Sophie Blat:** Writing – review & editing, Investigation. **Vincent Rioux:** Writing – review & editing, Supervision, Investigation, Funding acquisition, Conceptualization.

## Acknowledgments

The authors acknowledge the French dairy interbranch organization (CNIEL) for the financial support, ARCHE – BIOSIT platform for animal care, Daniel Catheline for his chemical expertise, Xavier Blanc (SAAJ-RAF INRAe, Jouy-en-Josas) for the elaboration of lipid free flour.

## Funding

This work was supported by the French dairy interbranch organization (CNIEL, Centre National Interprofessionel de l'Economie Laitière).

## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jnutbio.2024.109814.

## References

[1] Keys A, Aravanis C, Blackburn HW, Van Buchem FS, Buzina R, Djordjević BD, et al. Epidemiological studies related to coronary heart disease: characteristics of men aged 40-59 in seven countries. Acta Med Scand Suppl 1966;460:1-392. doi:10.1111/j.0954-6820.1966.tb04737.x.

[2] Siri-Tarino PW, Sun Q, Hu FB, Krauss RM. Meta-analysis of prospective cohort studies evaluating the association of saturated fat with cardiovascular disease.2013. Am J Clin Nutr 2010;91:535-46. doi:10.3945/ajcn.2009.27725.

[3] Legrand P, Rioux V. Specific roles of saturated fatty acids: beyond epidemiological data. Euro J Lipid Sci & Tech 2015;117:1489-99. doi:10.1002/ejlt.201400514.

[4] Fulco AJ. Fatty acid metabolism in bacteria. Prog Lipid Res 1983;22:133-60. doi:10.1016/0163-7827(83)90005-X.

[5] Kaneda T. Iso- and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance. Microbiol Rev 1991;55:288-302. doi:10.1128/mr.55.2.288-302.1991.

[6] Lindmark Månsson H. Fatty acids in bovine milk fat. Food Nutr Res 2008;52:1821. doi:10.3402/fnr.v52i0.1821.

[7] Brevik A, Veierød MB, Drevon CA, Andersen LF. Evaluation of the odd fatty acids 15:0 and 17:0 in serum and adipose tissue as markers of intake of milk and dairy fat. Eur J Clin Nutr 2005;59:1417-22. doi:10.1038/sj.ejcn.1602256.

[8] Wolk A, Vessby B, Ljung H, Barrefors P. Evaluation of a biological marker of dairy fat intake. Am J Clin Nutr 1998;68:291-5. doi:10.1093/ajcn/68.2.291.

[9] Imamura F, Fretts A, Marklund M, Ardisson Korat AV, Yang W-S, Lankinen M, et al. Fatty acid biomarkers of dairy fat consumption and incidence of type 2 diabetes: a pooled analysis of prospective cohort studies. PLoS Med 2018;15:e1002670. doi:10.1371/journal.pmed.1002670.

[10] Aune D, Norat T, Romundstad P, Vatten LJ. Dairy products and the risk of type 2 diabetes: a systematic review and dose-response meta-analysis of cohort studies. Am J Clin Nutr 2013;98:1066-83. doi:10.3945/ajcn.113.059030.

[11] Forouhi NG, Koulman A, Sharp SJ, Imamura F, Kröger J, Schulze MB, et al. Differences in the prospective association between individual plasma phospholipid saturated fatty acids and incident type 2 diabetes: the EPIC-InterAct case-cohort study. Lancet Diabetes Endocrinol 2014;2:810-18. doi:10.1016/S2213-8587(14)70146-9.

[12] Khaw K-T, Friesen MD, Riboli E, Luben R, Wareham N. Plasma phospholipid fatty acid concentration and incident coronary heart disease in men and women: the EPIC-Norfolk prospective study. PLoS Med 2012;9:e1001255. doi:10.1371/journal.pmed.1001255.

[13] Yoo W, Gjuka D, Stevenson HL, Song X, Shen H, Yoo SY, et al. Fatty acids in non-alcoholic steatohepatitis: focus on pentadecanoic acid. PLoS ONE 2017;12:e0189965. doi:10.1371/journal.pone.0189965.

[14] Wei W, Wong CC, Jia Z, Liu W, Liu C, Ji F, et al. Parabacteroides distasonis uses dietary inulin to suppress NASH via its metabolite pentadecanoic acid. Nat Microbiol 2023;8:1534-48. doi:10.1038/s41564-023-01418-7.

[15] Venn-Watson S, Lumpkin R, Dennis EA. Efficacy of dietary odd-chain saturated fatty acid pentadecanoic acid parallels broad associated health benefits in humans: could it be essential? Sci Rep 2020;10:8161. doi:10.1038/s41598-020-64960-y.

[16] Venn-Watson S. Pentadecanoylcarnitine is a newly discovered endocannabinoid with pleiotropic activities relevant to supporting physical and mental health. Sci Rep 2022;12:13717. doi:10.1038/s41598-022-18266-w.

[17] Weitkunat K, Schumann S, Nickel D, Hornemann S, Petzke KJ, Schulze MB, et al. Odd-chain fatty acids as a biomarker for dietary fiber intake: a novel pathway for endogenous production from propionate. Am J Clin Nutr 2017;105:1544-51. doi:10.3945/ajcn.117.152702.

[18] Burr GO, Burr MM. On the nature and rôle of the fatty acids essential in nutrition. J Biol Chem 1930;86:587-621. doi:10.1016/0163-7827(81)90004-7.

[19] Wesson LG, Burr GO. The metabolic rate and respiratory quotients of rats on a fat-deficient diet. J Biol Chem 1931;91:525-39. doi:10.1016/S0021-9258(18)76564-2.

[20] Choque B, Catheline D, Delplanque B, Guesnet P, Legrand P. Dietary linoleic acid requirements in the presence of α-linolenic acid are lower than the historical 2 % of energy intake value, study in rats. Br J Nutr 2015;113:1056-68. doi:10.1017/S0007114515000094.

[21] Burr GO, Burr MM. A 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.

[22] Menton DN. The effects of essential fatty acid deficiency on the fine structure of mouse skin. J Morphol 1970;132:181-205. doi:10.1002/jmor.1051320206.

[23] Mead JF, Slaton WH. Metabolism of essential fatty acids. J Biol Chem 1956;219:705-9. doi:10.1016/S0021-9258(19)63664-1.

[24] Guillocheau E, Drouin G, Catheline D, Orione C, Legrand P, Rioux V. Chemical synthesis and isolation of *trans*-palmitoleic acid (*Trans*-C16:1 n-7) suitable for nutritional studies. Euro J Lipid Sci & Tech 2020;122:1900409. doi:10.1002/ejlt.201900409.

[25] Astrup A, Bertram HC, Bonjour J-P, De Groot LC, De Oliveira Otto MC, et al. WHO draft guidelines on dietary saturated and trans fatty acids: time for a new approach? BMJ 2019;366:l4137. doi:10.1136/bmj.l4137.

[26] Guillocheau E, Garcia C, Drouin G, Richard L, Catheline D, Legrand P, et al. Retroconversion of dietary trans-vaccenic (trans-C18:1 n-7) acid to trans-palmitoleic acid (trans-C16:1 n-7): proof of concept and quantification in both cultured rat hepatocytes and pregnant rats. J Nutr Biochem 2019;63:19-26. doi:10.1016/j.jnutbio.2018.09.010.

[27] Drouin G, Guillocheau E, Catheline D, Baudry C, Le Ruyet P, Rioux V, et al. Impact of n-3 docosapentaenoic acid supplementation on fatty acid composition in rat differs depending upon tissues and is influenced by the presence of dairy lipids in the diet. J Agric Food Chem 2018;66:9976-88. doi:10.1021/acs.jafc.8b03069.

[28] Guillou H, D'Andrea S, Rioux V, Barnouin R, Dalaine S, Pedrono F, et al. Distinct
roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like
domain for rat Δ6-desaturase activity. J Lipid Res 2004;45:32–40. doi:10.1194/
jlr.M300339-JLR200.

[29] Blanchard H, Pédrono F, Boulier-Monthéan N, Catheline D, Rioux V, Legrand P. Comparative effects of well-balanced diets enriched in α-linolenic or linoleic acids on LC-PUFA metabolism in rat tissues. Prostaglandins Leukot Essent Fatty Acids 2013;88:383-9. doi:10.1016/j.plefa.2013.03.006.

[30] Pawlosky R, Sprecher H, Salem N. High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids. J Lipid Res 1992;33:1711-17. doi:10.1016/S0022-2275(20)41393-8.

[31] Bronsard J, Savary C, Massart J, Viel R, Moutaux L, Catheline D, et al. 3D multi-cell-type liver organoids: a new model of non-alcoholic fatty liver disease for drug safety assessments. Toxicol In Vitro 2024;94:105728. doi:10.1016/j.tiv.2023.105728.

[32] Launay Y, Jan I, Ciesielski V, Hue L, Succar M, Fret L, et al. Use of stable isotope-labeled fatty acids to measure desaturase activities with negative chemical ionization GC-MS. Chem Phys Lipids 2024;266:105451. doi:10.1016/j.chemphyslip.2024.105451.

[33] Bailes J, Soloviev M. Insulin-like growth factor-1 (IGF-1) and its monitoring in medical diagnostic and in sports. Biomolecules 2021;11:217. doi:10.3390/biom11020217.

[34] Laron Z. Insulin-like growth factor 1 (IGF-1): a growth hormone. Mol Pathol 2001;54:311-16. doi:10.1136/mp.54.5.311.

[35] Agoston DV. How to translate time? The temporal aspect of human and rodent biology. Front Neurol 2017;8:92. doi:10.3389/fneur.2017.00092.

[36] Likhar A, Patil MS. Importance of maternal nutrition in the first 1,000 days of life and its effects on child development: a narrative review. Cureus 2022;14(10):e30083. doi:10.7759/cureus.30083.

[37] Brody T. Vitamins. In: Nutritional biochemistry. San Diego: Academic Press; 1999. p. 491-692. doi:10.1016/B978-012134836-6/50012-3.

[38] Peluffo RO, Nervi AM, Brenner RR. Linoleic acid desaturation activity of liver microsomes of essential fatty acid deficient and sufficient rats. Biochim Biophys Acta 1976;441:25-31. doi:10.1016/0005-2760(76)90277-0.

[39] De Schrijver R, Privett OS. Hepatic fatty acids and acyl desaturases in rats: ef-
fects of dietary carbonhydrate and essential fatty acids. J Nutr 1983;113:2217-
22. doi:10.1093/jn/113.11.2217.

[40] Skrzypski J, Bellenger S, Bellenger J, Sinclair A, Poisson JP, Tessier C, et al. Re-
visiting delta-6 desaturase regulation by C18 unsaturated fatty acids, depend-
ing on the nutritional status. Biochimie 2009;91:1443-9. doi:10.1016/j.biochi.
2009.08.001.

[41] Nakamura MT, Nara TY. Structure, function, and dietary regulation of delta6, delta5, and delta9 desaturases. Annu Rev Nutr 2004;24:345-76. doi:10.1146/annurev.nutr.24.121803.063211.

[42] Legrand P, Catheline D, Hannetel JM, Lemarchal P. Stearoyl-CoA desaturase activity in primary culture of chicken hepatocytes. Influence of insulin, glucocorticoid, fatty acids and cordycepin. Int J Biochem 1994;26:777-85. doi:10.1016/0020-711X(94)90107-4.

[43] Shimano H, Yahagi N, Amemiya-Kudo M, Hasty AH, Osuga J, Tamura Y, et al. Sterol regulatory element-binding protein-1 as a key transcription factor for nutritional induction of lipogenic enzyme genes. J Biol Chem 1999;274:35832-9. doi:10.1074/jbc.274.50.35832.

[44] Legrand P, Catheline D, Rioux V, Durand G. Lauric acid is desaturated to 12:1n-3 by hepatocytes and rat liver homogenates. Lipids 2002;37:569-72. doi:10.1007/s11745-002-0934-y.

[45] Wang Z, Park HG, Wang DH, Kitano R, Kothapalli KSD, Brenna JT. Fatty acid desaturase 2 (FADS2) but not FADS1 desaturates branched chain and odd chain saturated fatty acids. Biochim Biophys Acta Mol Cell Biol Lipids 2020;1865:158572. doi:10.1016/j.bbalip.2019.158572.

[46] Kothapalli KSD, Park HG, Guo X, Sun X, Zou J, Hyon SS, et al. A novel FADS2 isoform identified in human milk fat globule suppresses FADS2 mediated Δ6-desaturation of omega-3 fatty acids. Prostaglandins, Leukot Essent Fatty Acids 2018;138:52-9. doi:10.1016/j.plefa.2018.06.004.

[47] Kothapalli KSD, Park HG, Kothapalli NSL, Brenna JT. FADS2 function at the major cancer hotspot 11q13 locus alters fatty acid metabolism in cancer. Prog Lipid Res 2023;92:101242. doi:10.1016/j.plipres.2023.101242.

[48] Vane JR, Bakhle YS, Botting RM. Cyclooxygenases 1 and 2. Annu Rev Pharmacol Toxicol 1998;38:97-120. doi:10.1146/annurev.pharmtox.38.1.97.

[49] Néglér R, Gaillard D, Ailhaud G. Prostacyclin as a potent effector of adipose-cell differentiation. Biochem J 1989;257:399-405. doi:10.1042/bj2570399.