Étienne Guillocheau

Philippe P Legrand

Vincent V Rioux
email: vincent.rioux@agrocampus-ouest.fr

Etienne Guillocheau

methods and chemical synthesis. A review

Keywords: Nutritional impacts, metabolism, origin, compositional data, analytical trans-palmitoleic acid, trans-vaccenic acid, rumenic acid, ruminant meat, ruminant milk, type 2 diabetes α-linolenic acid AOCS: American Oil Chemists' Society

Introduction 1.What is TPA?

Trans-palmitoleic acid (TPA) is a hexadecenoic fatty acid with one trans double bond located in the 9 th position using the Δ-nomenclature (trans-9-C16:1) (Figure 1A), or in the n-7 position using the Ω-nomenclature (trans-C16:1 n-7) (Figure 1B). It is worth stressing the importance of chemical details in this review. Indeed, TPA is usually confused in the literature with its cis isomer (cis-palmitoleic acid, cis-9-C16:1 or cis-C16:1 n-7, CPA), or with other trans-C16:1 positional isomers. Of note, TPA can also be quoted in the literature as "palmitelaidic acid". According to many reglementary definitions (e.g., Codex Alimentarius, European Food Standard Authority, Food and Drug Administration), TPA belongs to the trans fatty acid family [START_REF]Guidelines on Nutrition Labeling CAC/GL 2-1985 as Last Amended[END_REF][START_REF] Efsa | Opinion of the Scientific Panel on Dietetic Products , Nutrition and Allergies on a request from the Commission related to the presence of trans fatty acids in foods and the effect on human health of the consumption of trans fatty acids[END_REF][START_REF][END_REF], and more precisely to the trans-mono-unsaturated fatty acid (trans-MUFA) family.

Why focusing on TPA?

Why studying the physiological impacts of dietary TPA? The story began in 2008 when Cao and colleagues described a new lipokine corresponding to the fatty acid CPA [START_REF] Cao | Identification of a Lipokine, a Lipid Hormone Linking Adipose Tissue to Systemic Metabolism[END_REF][START_REF] Olefsky | Fat Talks, Liver and Muscle Listen[END_REF]. More precisely, the CPA coming from adipose tissue, and circulating as free fatty acid, was supposed to protect mice from hepatic de novo lipogenesis and insulin resistance [START_REF] Cao | Identification of a Lipokine, a Lipid Hormone Linking Adipose Tissue to Systemic Metabolism[END_REF]. Such a hypothesis is exciting, but hard to verify: the huge part of circulating CPA in humans arises from hepatic de novo lipogenesis, and not from adipose tissue [START_REF] Hodson | Is there something special about palmitoleate?[END_REF][START_REF] Mozaffarian | Circulating palmitoleic acid and risk of metabolic abnormalities and newonset diabetes[END_REF]. Alternatively, one could focus on TPA to better understand the physiological impacts of CPA [START_REF] Mozaffarian | Trans-palmitoleic acid, metabolic risk factors, and new-onset diabetes in U.S. adults[END_REF]. Mozaffarian and colleagues, therefore, assumed in 2010 that TPA would exert the same physiological impacts as CPA and would not be endogenously synthesized in humans [START_REF] Mozaffarian | Trans-palmitoleic acid, metabolic risk factors, and new-onset diabetes in U.S. adults[END_REF].

Explaining the whole story of TPA helps to understand the "magic" atmosphere that has always prevailed around this fatty acid. Before discovering the CPA as a lipokine, TPA was a barely known fatty acid that had just been reported in dairy products by a couple of studies [START_REF] Molkentin | Occurrence of trans-C16:1 acids in bovine milkfats and partially hydrogenated edible fats[END_REF][START_REF] Destaillats | Study of individual trans-and cis-16:1 isomers in cow, goat, and ewe cheese fats by gas-liquid chromatography with emphasis on the trans-Δ3 isomer[END_REF]. From this point of view, unravelling benefits TPA is clearly surprising, not to mention its belonging to the trans fatty acid family. Therefore, it is high time to make things clearer by gathering all knowledge about TPA.

Because there is often a confusion between TPA and its cis isomer in the literature, we shall review and talk about studies focusing only on TPA. The suggested benefits of TPA and potential mechanisms of action shall be discussed, along with its food sources. The very important metabolism of TPA shall be reviewed as well, to make connections with other ruminant (or natural) trans fatty acids (R-TFAs) that have been of scientific interest as well. Finally, we shall develop on topics related to TPA, especially about appropriate analytical methods.

Dietary TPA and health outcomes in humans

Benefits of dietary TPA acid arising from epidemiological data

Most knowledge about the physiological impact of dietary TPA comes from epidemiological data (Table 1). Prospective epidemiological studies highlighted a neutral role of dietary TPA on type 2 diabetes risk [START_REF] Kröger | Erythrocyte membrane phospholipid fatty acids, desaturase activity, and dietary fatty acids in relation to risk of type 2 diabetes in the European Prospective Investigation into Cancer and[END_REF][START_REF] Patel | Fatty acids measured in plasma and erythrocyte-membrane phospholipids and derived by food-frequency questionnaire and the risk of new-onset type 2 diabetes: A pilot study in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk c[END_REF][START_REF] Santaren | Serum pentadecanoic acid (15:0), a short-term marker of dairy food intake, is inversely associated with incident type 2 diabetes and its underlying disorders[END_REF]. Further, other prospective epidemiological data showed a significant inverse association between high levels of circulating TPA in humans and lower risk of type 2 diabetes [START_REF] Mozaffarian | Trans-palmitoleic acid, metabolic risk factors, and new-onset diabetes in U.S. adults[END_REF][START_REF] Yakoob | Circulating Biomarkers of Dairy Fat and Risk of Incident Diabetes Mellitus Among US Men and Women in Two Large Prospective Cohorts[END_REF][START_REF] Mozaffarian | trans-Palmitoleic acid, other dairy fat biomarkers, and incident diabetes: the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF]. Such an inverse relationship remained significant after adjustment on C15:0 [START_REF] Mozaffarian | trans-Palmitoleic acid, other dairy fat biomarkers, and incident diabetes: the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF] and C17:0 [START_REF] Mozaffarian | Trans-palmitoleic acid, metabolic risk factors, and new-onset diabetes in U.S. adults[END_REF], both fatty acids being also specific of dairy fat and suspected as well to have benefits on human health [START_REF] Pfeuffer | Pentadecanoic and Heptadecanoic Acids: Multifaceted Odd-Chain Fatty Acids[END_REF]. Therefore, TPA could certainly have special and unique benefits to human health. Importantly, two meta-analyses confirmed this significant and inverse relationship. Pooling different epidemiological studies, De Souza and colleagues highlighted a significant 42%-reduced risk of developing a type 2 diabetes associated with the highest levels of circulating TPA (mean risk ratio of 0.58, IC-95% = [0.58-0.74], P < 0.001) [START_REF] De Souza | Intake of saturated and trans unsaturated fatty acids and risk of all cause mortality, cardiovascular disease, and type 2 diabetes: systematic review and meta-analysis of observational studies[END_REF]. In agreement with this finding, Imamura and colleagues pooled several prospective analyses and found a significantly lower link between circulating levels of TPA in humans and type 2 diabetes incidence (mean hazard ratio of 0.82, IC-95% = [0.70-0.96], P < 0.05) [START_REF] Imamura | Fatty acid biomarkers of dairy fat consumption and incidence of type 2 diabetes: a pooled analysis of prospective cohort studies[END_REF].

Table 1 about here.

Regarding associations between circulating levels of TPA and anthropometric/metabolic parameters that might explain the decrease in type 2 diabetes risk, two prospective studies highlighted a better systemic insulin sensitivity (as measured by the HOMA-IR index) associated with high levels of circulating TPA [START_REF] Mozaffarian | Trans-palmitoleic acid, metabolic risk factors, and new-onset diabetes in U.S. adults[END_REF][START_REF] Mozaffarian | trans-Palmitoleic acid, other dairy fat biomarkers, and incident diabetes: the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF]. Of note, this higher insulin sensitivity seemed to be explained by a decreased fasting insulinemia rather than a decreased fasting glycemia [START_REF] Mozaffarian | Trans-palmitoleic acid, metabolic risk factors, and new-onset diabetes in U.S. adults[END_REF][START_REF] Mozaffarian | trans-Palmitoleic acid, other dairy fat biomarkers, and incident diabetes: the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF][START_REF] Da Silva | Natural Rumen-Derived trans Fatty Acids Are Associated with Metabolic Markers of Cardiac Health[END_REF]. However, better glucose uptake by the liver was pointed out as well to explain the lower systemic insulin resistance [START_REF] Yoo | Fatty acids in non-alcoholic steatohepatitis: Focus on pentadecanoic acid[END_REF][START_REF] Kratz | Dairy fat intake is associated with glucose tolerance, hepatic and systemic insulin sensitivity, and liver fat but not β-cell function in humans[END_REF]. These discrepancies underline the need in unravelling potential mechanisms of action of dietary TPA.

Epidemiological data are inconsistent as regards to cardiovascular risk. In the MESA cohort, high levels of circulating TPA had a neutral effect on cardiovascular disease [START_REF] De Oliveira Otto | Biomarkers of Dairy Fatty Acids and Risk of Cardiovascular Disease in the Multi-Ethnic Study of Atherosclerosis[END_REF]. Consistently, in the NHS and the HPFS cohorts, TPA was neutrally associated with risk of stroke [START_REF] Yakoob | Circulating biomarkers of dairy fat and risk of incident stroke in U.S. men and women in 2 large prospective cohorts[END_REF]. Three studies highlighted a favourable impact of dietary TPA on systolic blood pressure and hypertension [START_REF] Mozaffarian | trans-Palmitoleic acid, other dairy fat biomarkers, and incident diabetes: the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF][START_REF] De Oliveira Otto | Biomarkers of Dairy Fatty Acids and Risk of Cardiovascular Disease in the Multi-Ethnic Study of Atherosclerosis[END_REF][START_REF] Da Silva | Associations between dairy intake and metabolic risk parameters in a healthy French-Canadian population[END_REF], while two others reported a neutral effect [START_REF] Mozaffarian | Trans-palmitoleic acid, metabolic risk factors, and new-onset diabetes in U.S. adults[END_REF]. As regards to the impact on the total-cholesterol HDL-cholesterol , there is at least a neutral effect of dietary TPA [START_REF] Mozaffarian | trans-Palmitoleic acid, other dairy fat biomarkers, and incident diabetes: the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF] while a favourable effect was highlighted in the CHS cohort [START_REF] Mozaffarian | Trans-palmitoleic acid, metabolic risk factors, and new-onset diabetes in U.S. adults[END_REF]. Likewise, inconsistent outcomes are found concerning concentrations of LDL-cholesterol [START_REF] Mozaffarian | Trans-palmitoleic acid, metabolic risk factors, and new-onset diabetes in U.S. adults[END_REF][START_REF] Mozaffarian | trans-Palmitoleic acid, other dairy fat biomarkers, and incident diabetes: the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF][START_REF] De Oliveira Otto | Biomarkers of Dairy Fatty Acids and Risk of Cardiovascular Disease in the Multi-Ethnic Study of Atherosclerosis[END_REF] and HDL-cholesterol [START_REF] Mozaffarian | Trans-palmitoleic acid, metabolic risk factors, and new-onset diabetes in U.S. adults[END_REF][START_REF] Mozaffarian | trans-Palmitoleic acid, other dairy fat biomarkers, and incident diabetes: the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF][START_REF] De Oliveira Otto | Biomarkers of Dairy Fatty Acids and Risk of Cardiovascular Disease in the Multi-Ethnic Study of Atherosclerosis[END_REF][START_REF] Jacobs | Association between erythrocyte membrane fatty acids and biomarkers of dyslipidemia in the EPIC-Potsdam study[END_REF]. Overall, there is a need in supplementation studies specifically dedicated to the cardiovascular impact of dietary TPA.

Scarcity of TPA supplementation studies

The hypothesis of the benefits of dietary TPA on insulin resistance calls for supplementation studies that might unravel mechanisms of action. However, supplementation or nutritional studies involving TPA are quite scarce. The very low availability of pure TPA in enough amounts mainly explains this lack of data (see Section 6.2).

At the clinical level, to the best of our knowledge, only Lichtenstein and colleagues are currently carrying out a supplementation study focusing on probable benefits of dietary TPA towards insulin-resistance [START_REF] Lichtenstein | Palmitoleic Isomer Study[END_REF]. The situation is the same as regards to in vivo studies performed on rodents. Of note, a TPA-supplementation study carried on ApoE -/-male mice was recently published [START_REF] Cimen | Double bond configuration of palmitoleate is critical for atheroprotection[END_REF]. Given the chosen rodent model, this study dealt more about atherosclerosis rather than type 2 diabetes and insulin resistance. Indeed, TPA was found to be neutral as regards to the progression of atherosclerosis [START_REF] Cimen | Double bond configuration of palmitoleate is critical for atheroprotection[END_REF]. Besides, TPA did not impact the ratio between total cholesterol and HDL-cholesterol in plasma, suggesting a neutral effect of TPA on biomarkers of cardiovascular diseases [START_REF] Cimen | Double bond configuration of palmitoleate is critical for atheroprotection[END_REF].

To our knowledge, there is no supplementation study involving TPA on any rodent model dedicated to type 2 diabetes and insulin resistance. However, it is worth quoting that treatment of insulin-resistance by dietary TPA was patented in 2014 by Mozaffarian and colleagues [START_REF] Mozaffarian | Use of trans-palmitoleate in identifying and treating metabolic disease[END_REF]. Few details are available in this patent, but several results can be found. On the one hand, C2C12 myotubes cultured in the presence of TPA had a higher uptake of extracellular glucose compared with both control and palmitic acid treatment [START_REF] Mozaffarian | Use of trans-palmitoleate in identifying and treating metabolic disease[END_REF]. This result suggests a direct impact of dietary TPA on glycemia, which is in line with the conclusions of Kratz and colleagues [START_REF] Kratz | Dairy fat intake is associated with glucose tolerance, hepatic and systemic insulin sensitivity, and liver fat but not β-cell function in humans[END_REF]. However, this result is contrary to epidemiological studies that pointed out a likely role of dietary TPA on fasting insulinemia rather than on fasting glycemia [START_REF] Mozaffarian | Trans-palmitoleic acid, metabolic risk factors, and new-onset diabetes in U.S. adults[END_REF][START_REF] Mozaffarian | trans-Palmitoleic acid, other dairy fat biomarkers, and incident diabetes: the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF][START_REF] Da Silva | Natural Rumen-Derived trans Fatty Acids Are Associated with Metabolic Markers of Cardiac Health[END_REF]. On the other hand, 200 mg/kg body weight TPA supplementation on mice (presumably both Ob/Ob and healthy C57BL/6 mice fed a normal and a high-fat diet) were carried out and confirmed that TPA could be incorporated in plasma free fatty acids, phospholipids and triglycerides [START_REF] Mozaffarian | Use of trans-palmitoleate in identifying and treating metabolic disease[END_REF].

No further details about other outcomes are provided.

To unravel potential mechanisms of action of dietary TPA, one should rely on a couple of in vitro studies. First, compared with dietary oleic acid, TPA did not impact insulin secretion on perfused rat pancreas. However, TPA stimulated insulin secretion to a higher extent than palmitic acid [START_REF] Stein | The insulinotropic potency of fatty acids is influenced profoundly by their chain length and degree of saturation[END_REF]. In a straight relationship with these results, TPA rose PPAR-γ activity and pancreatic and duodenal homeobox 1 (PDX-1) in pancreatic β INS-1 cells [START_REF] Kraft | Dairy-derived bioactive fatty acids improve pancreatic β-cell function[END_REF]. Taken together, these in vitro outcomes are in line with epidemiological studies which highlighted a lower insulin secretion in subjects having high circulating levels of TPA. Pancreas might, therefore, be a target for dietary TPA to exert its benefits. Second, on endothelial HUVEC cells, TPA led to a significant decrease in the expression of tumor necrosis factor-α (TNF-α), vascular cell adhesion molecule 1 (VCAM-1) and superoxide-dismutase-2 (SOD-2) [START_REF] Da Silva | Trans fatty acids suppress TNFα-induced inflammatory gene expression in endothelial (HUVEC) and hepatocellular carcinoma (HepG2) cells[END_REF]. Likewise, TNF-α was significantly downshifted in hepatic HepG2 cells incubated with TPA [START_REF] Da Silva | Trans fatty acids suppress TNFα-induced inflammatory gene expression in endothelial (HUVEC) and hepatocellular carcinoma (HepG2) cells[END_REF]. In both HUVEC and HepG2 cells, these outcomes were independent of PPAR-γ activation. Thus, the anti-inflammatory effect of dietary TPA is suggested, in agreement with epidemiological data. However, such outcomes were found neither on healthy human endothelial cells nor on diabetic human ones [START_REF] Livingstone | Comparative effect of dairy fatty acids on cell adhesion molecules, nitric oxide and relative gene expression in healthy and diabetic human aortic endothelial cells[END_REF].

Explaining the circulating levels of TPA in humans: dietary TPA as a direct origin

Circulating levels of TPA in humans can be explained by its dietary origin, as TPA is found in several foods. Because TPA arises from ruminal biohydrogenation, ruminant-derived foods such as ruminant milk and ruminant meat do contain detectable amounts of TPA. Since TPA is a trans fatty acid, there is also evidence of its presence in partially hydrogenated oils.

Ruminant-derived foods as the only forthcoming source of TPA

Epidemiological data pointed out that ruminant-derived foods are among the sources of dietary TPA (Table 2). First, cross-sectional studies highlighted a positive correlation between intakes of dairy products and circulating TPA in humans [START_REF] Da Silva | Associations between dairy intake and metabolic risk parameters in a healthy French-Canadian population[END_REF][START_REF] Nestel | Specific plasma lipid classes and phospholipid fatty acids indicative of dairy food consumption associate with insulin sensitivity[END_REF][START_REF] Pranger | Circulating fatty acids as biomarkers of dairy fat intake: Data from the Lifelines Biobank and Cohort Study[END_REF]. Second, studies carried out at the prospective level also confirmed the positive association between ruminant-derived foods and circulating levels of TPA in humans. In the NHS cohort, levels of TPA in both plasma and RBC were significantly and positively correlated to dairy fat consumption [START_REF] Sun | Plasma and erythrocyte biomarkers of dairy fat intake and risk of ischemic heart disease[END_REF]. Micha and colleagues reported a significant contribution of both dairy products and red meat in explaining circulating levels of TPA in the CHS cohort [START_REF] Micha | Food sources of individual plasma phospholipid trans fatty acid isomers: the Cardiovascular Health Study[END_REF]. In strong agreement with these findings, circulating levels of TPA were significantly explained by both red meat and dairy product consumption in the MESA cohort [START_REF] De Oliveira Otto | Biomarkers of Dairy Fatty Acids and Risk of Cardiovascular Disease in the Multi-Ethnic Study of Atherosclerosis[END_REF]. Positive and significant correlations between ruminantderived foods and circulating levels of TPA were also found in the HPFS cohort [START_REF] Yakoob | Circulating Biomarkers of Dairy Fat and Risk of Incident Diabetes Mellitus Among US Men and Women in Two Large Prospective Cohorts[END_REF][START_REF] Yakoob | Circulating biomarkers of dairy fat and risk of incident stroke in U.S. men and women in 2 large prospective cohorts[END_REF]. Finally, a recent meta-analysis confirmed that dietary TPA stems from consumption of dairy products [START_REF] Pranger | Fatty acids as biomarkers of total dairy and dairy fat intakes: a systematic review and meta-analysis[END_REF].

Table 2 about here.

As TPA is a naturally occurring trans fatty acid, one should look at ruminal biohydrogenation to explain its presence in ruminant-derived foods. The workflow of the ruminal biohydrogenation mainly starts with the dietary linoleic (C18:2 n-6, LA) and αlinolenic (C18:3 n-3, ALA) acids, and ends with the stearic acid (C18:0). R-TFAs with 18 carbons arise as intermediates of this ruminal biohydrogenation. Because ruminal biohydrogenation involves a wide range of bacteria species and metabolic pathways, the diversity of R-TFAs is high. As such, R-TFAs include isomers of trans-C18:1 acids, conjugated linoleic acids (CLA), conjugated linolenic acids (CLnA) and C18:2 fatty acids with one trans double bond, as previously reviewed [START_REF] Ferlay | Production of trans and conjugated fatty acids in dairy ruminants and their putative effects on human health: A review[END_REF][START_REF] Enjalbert | Biosynthesis of trans fatty acids in ruminants[END_REF][START_REF] Chilliard | Diet, rumen biohydrogenation and nutritional quality of cow and goat milk fat[END_REF]. Importantly, very accurate metabolic pathways are naturally favoured in the rumen. Thus, trans-vaccenic acid (trans-11 C18:1, or trans-C18:1 n-7, TVA) is the main fatty acid among the trans-C18:1 positional isomers naturally generated [START_REF] Wolff | Improvement in the resolution of individual trans-18:1 isomers by capillary gas-liquid chromatography: use of a 100-m CP-Sil 88 column[END_REF]. Likewise, rumenic acid (cis-9, trans-11 C18:2, RMA) is the main isomer among the CLA isomers naturally generated by ruminal biohydrogenation [START_REF] Sehat | Identification of conjugated linoleic acid isomers in cheese by gas chromatography, silver ion high performance liquid chromatography and mass spectral reconstructed ion profiles. Comparison of chromatographic elution sequences[END_REF].

TPA being a trans C16:1 fatty acid, where does it stem from? So far, two hypotheses may explain the presence of TPA in ruminant-derived foods (Figure 2). First, chain-shortening of TVA might occur. Such a pathway has been proposed for a long time to explain the presence of TPA in ruminant milk [START_REF] Luna | Occurrence of C16:1 isomers in milk fats from ewes fed with different dietary lipid supplements[END_REF][START_REF] Hay | Isomeric monoenoic fatty acids in bovine milk fat[END_REF]. Second, TPA might also arise from the ruminant biohydrogenation of dietary C16:3 n-3 fatty acid (C16:3 7-cis,10-cis,13-cis) [START_REF] Destaillats | Study of individual trans-and cis-16:1 isomers in cow, goat, and ewe cheese fats by gas-liquid chromatography with emphasis on the trans-Δ3 isomer[END_REF] which occurs in "C16:3 plants" [START_REF] Dörmann | Membrane lipids[END_REF][START_REF] Leray | Les lipides dans le monde vivant: introduction à la lipidomique[END_REF]. Assuming a similar workflow to that of ALA biohydrogenation, TPA can indeed be obtained from C16:3 n-3 (Figure 3) [START_REF] Destaillats | Study of individual trans-and cis-16:1 isomers in cow, goat, and ewe cheese fats by gas-liquid chromatography with emphasis on the trans-Δ3 isomer[END_REF]. In fact, both hypotheses might be true, so that TPA in ruminant milk would be explained by two metabolic pathways. Of note, the involvement of dietary C16:2 n-6 fatty acid in TPA synthesis in ruminants was also assumed similarly to biohydrogenation of dietary LA [START_REF] Kramer | Combining results of two GC separations partly achieves determination of all cis and trans 16:1, 18:1, 18:2 and 18:3 except CLA isomers of milk fat as demonstrated using Ag-ion SPE fractionation[END_REF], but this hypothesis has remained unexplored so far. When it comes to human nutrition, ruminant meat and milk are of high interest. TPA generated through ruminal biohydrogenation may enter the general circulation of the ruminant and be incorporated in muscle and mammary gland. Because fatty acids including trans ones can be metabolised, another question arises about chain-shortening of TVA: does peroxisomal β-oxidation occur in the rumen, in ruminant tissues, or in both? So far, this question remains unresolved but peroxisomal β-oxidation is very likely to occur in ruminant tissues. On the contrary, there are uncertainties about chain-shortening of TVA in the rumen (Figure 2).

Reliable analytical data confirm the presence of TPA in ruminant-derived foods available at retail. Regarding ruminant milk, only Destaillats and colleagues reported very reliable TPA contents in ruminant milk available at retail in 2000, based on silver-ion thin-layer chromatography (Ag + -TLC) and appropriate gas-chromatography (GC) analysis [START_REF] Destaillats | Study of individual trans-and cis-16:1 isomers in cow, goat, and ewe cheese fats by gas-liquid chromatography with emphasis on the trans-Δ3 isomer[END_REF]. Regardless of the ruminant species, TPA was the major trans-C16:1 positional isomer and accounts for 31.7, 26.6 and 31.1% of total trans-C16:1 in cow, goat and ewe milk available at retail, respectively [START_REF] Destaillats | Study of individual trans-and cis-16:1 isomers in cow, goat, and ewe cheese fats by gas-liquid chromatography with emphasis on the trans-Δ3 isomer[END_REF]. Our research group also found similar relative levels of TPA (as % of total trans-C16:1) in ruminant milk available in France in 2018 [START_REF] Guillocheau | Dairy products as main dietary sources of trans-palmitoleic and trans-vaccenic acid in France[END_REF] (Figure 4A). It is worth reminding that having several positional trans-C16:1 isomers is compatible with having multiple ruminal biohydrogenation pathways, including one being naturally favoured and leading to TPA. As regards to absolute concentrations, TPA accounted for 54, 41 and 80 mg/100 g of total fatty acids for cow, goat and ewe milk, respectively [START_REF] Destaillats | Study of individual trans-and cis-16:1 isomers in cow, goat, and ewe cheese fats by gas-liquid chromatography with emphasis on the trans-Δ3 isomer[END_REF], while total trans-C16:1 fatty acids accounted for 0.13% (± 0.05, standard deviation) of total fatty acids in cow milk available at retail [START_REF] Molkentin | Occurrence of trans-C16:1 acids in bovine milkfats and partially hydrogenated edible fats[END_REF].

As for ruminant meat available at retail, to the best of our knowledge, our research group was the very first to report accurate content in TPA, based on reliable analytical methods [START_REF] Guillocheau | Dairy products as main dietary sources of trans-palmitoleic and trans-vaccenic acid in France[END_REF]. Like ruminant milk available at retail, TPA was the major trans-C16:1 isomer accounting for approx. 40% of total trans-C16:1 (Figure 4B) and weighted 0.03% of total fatty acids [START_REF] Guillocheau | Dairy products as main dietary sources of trans-palmitoleic and trans-vaccenic acid in France[END_REF].

It is interesting to note a similar pattern between TPA, TVA and RMA in ruminant products. More precisely, TPA, TVA and RMA are the major positional isomers among trans-C16:1, trans-C18:1 and CLA, respectively. Such a result may be interpreted in two ways. First, it may reflect the metabolic links connecting trans-C16:1, trans-C18:1 and CLA between each other, once these fatty acids are incorporated in tissues of ruminant species. As a result, one can easily understand the closeness between the trans-C16:1, trans-C18:1 and CLA profiles in ruminantderived products. Second, it may also highlight that accurate metabolic pathways are favoured when it comes to ruminal biohydrogenation of dietary C16:3 n-3, yielding TPA as the major trans-C16:1 isomer. In this case, these metabolic pathways are the same as those yielding TVA and RMA among the trans-C18:1 and CLA, respectively.

Partially hydrogenated oils as a former source of TPA

Given that TPA is a trans-MUFA, the link with industrial processes that generate such fatty acids seems logical. Several industrial processes have a well-known ability to generate such trans fatty acids: partial (or catalytic) hydrogenation, deodorization and deep-frying. Again, most knowledge about trans-MUFAs of industrial origin is related to trans-C18:1 positional isomers. On the contrary, barely anything is known about trans-C16:1 positional isomers, including TPA.

Deodorization of crude oils generate trans-PUFAs, but not any trans-MUFA (i.e., neither trans-C18:1 nor trans-C16:1 positional isomers) [START_REF] Bezelgues | Formation of trans fatty acids during deodorization of edible oils[END_REF][START_REF] Ratnayake | Analysis of dietary trans fatty acids[END_REF]. It is now a process in which the formation of trans-PUFAs is highly controlled [START_REF] Bezelgues | Formation of trans fatty acids during deodorization of edible oils[END_REF]. As for deep-frying, studies carried out in the 1990s did underline the formation of trans-MUFAs (trans-C18:1 and presumably trans-C16:1) [START_REF] Ovesen | Fatty acid composition and contents of trans monounsaturated fatty acids in frying fats, and in margarines and shortenings marketed in Denmark[END_REF], but at the current time, no trans-MUFAs (neither trans-C18:1 nor trans-C16:1 positional isomers) are reported following this step [START_REF] Kuhnt | Trans fatty acid isomers and the trans-9/trans-11 index in fat containing foods[END_REF][START_REF] Astiasarán | Margarines and Fast-Food French Fries: Low Content of trans Fatty Acids[END_REF][START_REF] Chen | The analysis of trans fatty acid profiles in deep frying palm oil and chicken fillets with an improved gas chromatography method[END_REF]. Partial (or catalytic) hydrogenation has been applied for several decades to crude oils, yielding the famous partially hydrogenated oils (PHOs). During this process, the cis ethylenic double bond of fatty acid (cis-MUFAs or cis-PUFAs) can be isomerised into a trans double bond and can also change of position across the carbon chain [START_REF] Bezelgues | Formation of trans fatty acids during hydrogenation of edible oils[END_REF]. Here, two cases should be distinguished: partially hydrogenated vegetable oils (PHVOs), and partially hydrogenated fish oils (PHFOs).

Partially hydrogenated vegetable oils: a lack of reliable data and the question of their current use

Several epidemiological studies suggest the presence of TPA in PHVOs (Table 2). In 2002, Baylin and colleagues highlighted a significant and positive correlation between TPA content in human subcutaneous adipose tissue and vegetable fat consumption in Costa Rica [START_REF] Baylin | Adipose tissue biomarkers of fatty acid intake[END_REF]. Consistently in the MESA cohort, consumption of margarine and salted biscuits significantly contributed to circulating levels of TPA [START_REF] De Oliveira Otto | Biomarkers of Dairy Fatty Acids and Risk of Cardiovascular Disease in the Multi-Ethnic Study of Atherosclerosis[END_REF]. Finally, a significant and positive link was found between PHVO consumption and circulating levels of TPA in the IRAS cohort [START_REF] Santaren | Serum pentadecanoic acid (15:0), a short-term marker of dairy food intake, is inversely associated with incident type 2 diabetes and its underlying disorders[END_REF]. These results underline that ruminant-derived foods may not be the only contributors to TPA intakes in humans and underline the need for reliable data about trans-C16:1 positional isomers in PHVOs.

CPA is commonly found in crude vegetable oils, accounting for a couple of % of total fatty acids depending on the vegetable oil [START_REF] Hodson | Is there something special about palmitoleate?[END_REF]. From a theoretical point of view, getting TPA after partial hydrogenation of such oils does make sense. Qualitatively speaking, the situation might be the same as for the trans-C18:1 profile in PHVOs, which is characterized by a wide range of positional isomers in almost equal amounts [START_REF] Wolff | Follow-up of the ∆4 to ∆16 trans-18:1 isomer profile and content in French processed foods containing partially hydrogenated vegetable oils during the period 1995-1999. Analytical and nutritional implications[END_REF]. As a result, the theoretical trans-C16:1 profile should contain TPA plus other trans-C16:1 positional isomers in similar amounts. The amount of trans-C16:1 should, however, be way lower than that of trans-C18:1 fatty acids. However, to the best of our knowledge, no reliable data exist so far about trans-C16:1 positional isomers in PHVOs. Dietz Precht and Joachim Molkentin previously attempted to do so, but the content in trans-C16:1 in such oils was too low for allowing an accurate resolution of trans-C16:1 positional isomers and assessing TPA content [START_REF] Molkentin | Occurrence of trans-C16:1 acids in bovine milkfats and partially hydrogenated edible fats[END_REF][START_REF] Precht | Recent trends in the fatty acid composition of German sunflower margarines, shortenings and cooking fats with emphasis on individual C16:1[END_REF]. Nonetheless, total trans-C16:1 were accurately and reliably quantified in PHVOs, based on exactly the same analytical methods as that of Destaillats and colleagues [START_REF] Destaillats | Study of individual trans-and cis-16:1 isomers in cow, goat, and ewe cheese fats by gas-liquid chromatography with emphasis on the trans-Δ3 isomer[END_REF]. Commercially available German PHVOs in 1997 had a mean content of trans-C16:1 equal to 0.01% of total fatty acids (± 0.03, standard deviation) [START_REF] Molkentin | Occurrence of trans-C16:1 acids in bovine milkfats and partially hydrogenated edible fats[END_REF]. In the early 2000s, still in Germany, total trans-C16:1 accounted for 0.01% of total fatty acids (± 0.01, standard deviation) in sunflower margarines and 0.01% (± 0.02, standard deviation) in cooking fats/shortenings available at retail [START_REF] Precht | Recent trends in the fatty acid composition of German sunflower margarines, shortenings and cooking fats with emphasis on individual C16:1[END_REF]. Taken together, these data confirm that very low amounts of trans-C16:1 are found in PHVOs. Based on this quantitative data, plus on the trans-C16:1 content in German maternal milk, it was concluded that PHVOs could not be deemed as a reliable contributor to TPA intakes, contrary to dairy products [START_REF] Precht | Identification and quantitation of cis/trans C16:1 and C17:1 fatty acid positional isomers in German human milk lipids by thin-layer chromatography and gas chromatography/mass spectrometry[END_REF].

Despite accurate analytical data, the hypothesis according to which PHVOs contribute to TPA intakes seems plausible. Indeed, most epidemiological data rely on blood sampling carried out in the 1990s (Table 2). At that time, PHVOs were still used in a wide range of foods. Therefore, such epidemiological data cannot be used to claim with certainty that TPA is a biomarker of ruminant-derived food consumption. But right now, the main question is whether PHVOs are still in use nowadays at least in Western countries. At the regulation level, countries such as the USA [START_REF][END_REF], Canada [START_REF]Gazette du Canada, Règlement modifiant le règlement sur les aliments et les drogues (étiquetage nutritionnel, allégations relatives à la teneur nutritive et allégations relatives à la santé)[END_REF] and Denmark [START_REF] Stender | The influence of trans fatty acids on health[END_REF] were trailblazers in the early 2000s, taking the very first steps to progressively decrease the use of PHVOs in foods. In 2018, both the USA and Canada settled a ban on PHVOs [START_REF]Gazette du Canada, Règlement modifiant certains règlements pris en vertu de la Loi sur les aliments et drogues (symboles nutritionnels, autres dispositions d'étiquetage, huiles partiellement hydrogénées et vitamine D)[END_REF][START_REF] Fda | Final determination regarding partially hydrogenated oils[END_REF]. The European Commission has recently adopted a legal 2%-limit of industrial trans fatty acids in all foods which should enter into force by April 2021 [64]. Such steps clearly shed light on the PHVO issue which received high media coverage and encouraged industries to look for alternative solutions (i.e., the voluntary reduction approach). Both the regulation steps and the voluntary reduction approach led to decreased levels of industrial trans fatty acids, which are now barely detectable in foods [START_REF] Astiasarán | Margarines and Fast-Food French Fries: Low Content of trans Fatty Acids[END_REF][START_REF] Ansorena | No trans fatty acids in Spanish bakery products[END_REF][START_REF] Costa | Trans fatty acids in the Portuguese food market[END_REF][START_REF] Santos | Trans fatty acids in commercial cookies and biscuits: An update of Portuguese market[END_REF][START_REF] Roe | Trans fatty acids in a range of UK processed foods[END_REF][START_REF] Kroustallaki | Fatty acid composition of Greek margarines and their change in fatty acid content over the past decades[END_REF]. This trend is confirmed by epidemiological data highlighting a decrease in trans-C18:1 content in human plasma and red blood cells, as reviewed by Craig-Schmidt and colleagues [START_REF] Craig-Schmidt | Evolution of worldwide consumption of trans fatty acids[END_REF]. However, recent studies underlined a boundary between Western European countries and Eastern ones, the latter still having elevated amounts of industrial trans fatty acids compared to the former [START_REF] Stender | A trans European Union difference in the decline in trans fatty acids in popular foods: a market basket investigation[END_REF][START_REF] Stender | Tracing artificial trans fat in popular foods in Europe: a market basket investigation[END_REF][START_REF] Stender | Artificial trans fat in popular foods in 2012 and in 2014: a market basket investigation in six European countries[END_REF][START_REF] Stender | Industrially produced trans fat in popular foods in 15 countries of the former Soviet Union from 2015 to 2016: a market basket investigation[END_REF]. Thus, further research is therefore needed to conclude about the current use of PHVOs and its contribution to TPA intakes by analysing bakery foods and biscuits.

Partially hydrogenated fish oils: a former and geographically restricted source of TPA

In addition to the application on vegetable oils, partial hydrogenation can also be applied to crude fish oils to yield PHFOs. Crude fish oils do contain CPA which accounts for 7% of total fatty acids in cod liver oil [START_REF] Hodson | Is there something special about palmitoleate?[END_REF]. As a result, partial hydrogenation of crude fish oils should theoretically lead to TPA plus plenty of other positional trans-C16:1 isomers.

To the best of our knowledge, there is no epidemiological data linking PHFO consumption to circulating levels of TPA in humans. Instead, very accurate data exist concerning the trans-C16:1 positional isomers in PHFOs. TPA was the major trans-C16:1 positional isomer in German cooking fats containing PHFOs and available at retail at the end of the 1990s, and weighted 24% of total trans-C16:1 in these oils [START_REF] Molkentin | Occurrence of trans-C16:1 acids in bovine milkfats and partially hydrogenated edible fats[END_REF]. Other trans-C16:1 positional isomers, especially trans-6-8 C16:1 and trans-10-C16:1, weighed almost as much as TPA [START_REF] Molkentin | Occurrence of trans-C16:1 acids in bovine milkfats and partially hydrogenated edible fats[END_REF]. Similar patterns in PHFOs were obtained in two other studies [START_REF] Mjøs | Trans fatty acid analyses in samples of marine origin: The risk of false positives[END_REF][START_REF] Kraft | Silver ion solid-phase extraction chromatography for the analysis of trans fatty acids[END_REF] and by our research group [START_REF] Guillocheau | Using GC-MS and helium to resolve positional isomers of trans-C16:1 and trans-C18:1 fatty acids[END_REF]. It is worth underlining that all these studies relied on appropriate analytical methods, thus generating reliable data. Regarding absolute concentrations, trans-C16:1 in PHFOs accounted for 1.89% of total fatty acids (± 0.63, standard deviation) in Germany in 1997 [START_REF] Molkentin | Occurrence of trans-C16:1 acids in bovine milkfats and partially hydrogenated edible fats[END_REF].

Given the complexity of the fatty acid composition of crude fish oils, one may also assume that TPA can naturally be found in crude fish oils. To our knowledge, only Fardin-Kia and colleagues analysed with high accuracy trans-C16:1 positional isomers in crude menhaden oil and highlighted the presence of only trans-6-C16:1 but not TPA [START_REF] Fardin-Kia | Separation of the fatty acids in menhaden oil as methyl esters with a highly polar ionic liquid gas chromatographic column and identification by time of flight mass spectrometry[END_REF]. Thus, only partial hydrogenation can explain the presence of TPA and other trans-C16:1 positional isomers (excepted the trans-6-C16:1) in PHFOs.

Because of these high amounts of TPA in PHFOs, such oils presumably contributed to TPA intakes in combination with dairy products in Germany in the late 1990s [START_REF] Precht | Identification and quantitation of cis/trans C16:1 and C17:1 fatty acid positional isomers in German human milk lipids by thin-layer chromatography and gas chromatography/mass spectrometry[END_REF]. This conclusion suggests that PHFOs were still used in Germany at that time. In fact, little information exists about the use of these oils. Importantly, PHFOs are described as excellent alternatives to PHVOs when it comes to the production of shortenings, cooking fats and margarines [START_REF] Iafmm | The Substitutability of P.H. Fish Oils Vegetable Oils and Animal Fats[END_REF][START_REF] Van Erp-Baart | Trans fatty acids in bakery products from 14 European countries: The TRANSFAIR study[END_REF][START_REF] Young | The refining and hydrogenation of fish oil[END_REF]. Still, the use of PHFOs was likely to be restricted to European countries [START_REF] Young | The Usage of Hydrogenated Fish Oils in Maragarines, Shortenings and Compound Fats[END_REF], and more precisely Northern European countries like Germany, Netherlands, the United Kingdom or Norway [START_REF] Wolff | Follow-up of the ∆4 to ∆16 trans-18:1 isomer profile and content in French processed foods containing partially hydrogenated vegetable oils during the period 1995-1999. Analytical and nutritional implications[END_REF][START_REF] Laake | A prospective study of intake of trans-fatty acids from ruminant fat, partially hydrogenated vegetable oils, and marine oils and mortality from CVD[END_REF][START_REF] Mensink | Trans monounsaturated fatty acids in nutrition and their impact on serum lipoprotein levels in man[END_REF]. In addition, there is evidence about an end of use and consumption of PHFOs in these countries in the late 1990s (Bente Kirkhus, NOFIMA, personal communication). Thus, PHFOs were a strong but former contributor of TPA dietary intakes in Northern European countries and did not impact on epidemiological studies that were conducted in the USA.

Explaining the circulating levels of TPA in humans: endogenous retro-conversion of dietary TVA as an indirect origin

Evidence of endogenous retro-conversion of dietary TVA to TPA

Generally speaking, trans-C16:1 fatty acids in non-ruminants have always been hypothesized to arise from their dietary trans-C18:1 counterparts. Rats fed a PHVO containing trans-C18:1 but deprived of trans-C16:1 isomers had detectable levels of trans-C16:1 isomers in lipid classes of the liver [START_REF] Chumbler | Incorporation Octadecenoate of Dietary cis and trans Isomers of in Lipid Classes of Liver and Hepatoma[END_REF][START_REF] Wolff | Evolution au cours du temps de la composition en chaînes alkényles et acyles des plasmalogènes de mitochondries de coeur et de reins chez des rats ingérant de la triélaïdine[END_REF][START_REF] Wood | Incorporation of Dietary cis and trans Octadecenoate Isomers in the Lipid Classes of Various Rat Tissues[END_REF]. Recently, Jaudzsus and colleagues reported an increase in TPA content in human red blood cells following a TVA supplementation [START_REF] Jaudszus | trans Palmitoleic acid arises endogenously from dietary vaccenic acid[END_REF]. It is interesting to note that such a hypothesis corresponds to one assumption that is made to explain the origin of TPA in ruminant milk and meat. Consistently, adding TVA to the culture medium of RINm5F cells led to the detection of TPA in these cells [START_REF] Sarnyai | Cellular toxicity of dietary trans fatty acids and its correlation with ceramide and diglyceride accumulation[END_REF].

Our research group accurately demonstrated that TVA could indeed undergo one cycle of peroxisomal β-oxidation, yielding TPA [START_REF] Guillocheau | 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[END_REF]. We first demonstrated that the growing concentrations of TVA in the culture medium of cultured rat hepatocytes led to growing amounts of intracellular TPA, but also to growing amounts of TPA in secreted triglycerides of these hepatocytes [START_REF] Guillocheau | 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[END_REF]. The conversion rate was estimated at 10%, in accordance with the assumptions of Jaudszus and colleagues [START_REF] Jaudszus | trans Palmitoleic acid arises endogenously from dietary vaccenic acid[END_REF]. It is worth underlining that we formally characterized TPA by a combination of Ag + -TLC, appropriate GC analysis and DMOX derivatives. Second, we highlighted that specific blockade of peroxisomal β-oxidation in these hepatocytes significantly decreased the amount of intracellular TPA, but not specific blockade of mitochondrial β-oxidation [START_REF] Guillocheau | 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[END_REF]. Third, pregnant rats fed either a low-TVA (0.1% of total energy) or a high-TVA diet (4% of total energy) had detectable levels of TPA in the liver, accounting for 0.02% and 0.11% of total fatty acids, respectively [START_REF] Guillocheau | 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[END_REF]. Taken together, our findings demonstrated that dietary TVA can undergo peroxisomal β-oxidation yielding TPA. The liver is very likely to ensure a great part of this retro-conversion, and the subsequent TPA can be exported to other tissues through secreted triglycerides.

Dietary sources of TVA: the same as dietary sources of TPA

In addition to dietary sources of TPA, one should therefore also look at dietary sources of TVA to explain circulating levels of TPA in humans. At the epidemiological level and of course considering solely TVA and not total trans-C18:1 fatty acids, there is to our knowledge only the Lifelines Biobank and Cohort Study which concluded that dietary TVA was supplied by dairy products in the Netherlands in the late 2000s [START_REF] Pranger | Circulating fatty acids as biomarkers of dairy fat intake: Data from the Lifelines Biobank and Cohort Study[END_REF][START_REF] Pranger | Potential Biomarkers for Fat from Dairy and Fish and Their Association with Cardiovascular Risk Factors : Cross-sectional Data from the LifeLines Biobank and Cohort Study[END_REF]. This result is consistent with what was said above. Indeed, TVA typically arises as an intermediate of ruminal biohydrogenation, and more precisely as the major trans-C18:1 isomer. Accordingly, our research group found that TVA accounted for the major trans-C18:1 isomer in a range of commercially available French dairy products, weighing approx. 50% of total trans-C18:1 fatty acids (Figure 5A) [START_REF] Guillocheau | Dairy products as main dietary sources of trans-palmitoleic and trans-vaccenic acid in France[END_REF]. As regards to absolute concentrations, TVA accounted for approx. 1.8% of total fatty acids [START_REF] Guillocheau | Dairy products as main dietary sources of trans-palmitoleic and trans-vaccenic acid in France[END_REF]. Both our qualitative and quantitative data are in good agreement with previous accurate results obtained from dairy products commercially available in France, Germany, Canada and Japan [START_REF] Precht | Comparative studies on individual isomeric 18:1 acids in cow, goat, and ewe milk fats by low-temperature high-resolution capillary gas-liquid chromatography[END_REF][START_REF] Wolff | Content and distribution of trans-18:1 acids in ruminant milk and meat fats. Their importance in european diets and their effect on human milk[END_REF][START_REF] Molkentin | Optimized analysis of trans-octadecenoic acids in edible fats[END_REF][START_REF] Precht | Rapid analysis of the isomers of trans-octadecenoic acid in milk fat[END_REF][START_REF] Mendis | Fatty Acid Profile of Canadian Dairy Products with Special Attention to the trans-Octadecenoic Acid and Conjugated Linoleic Acid Isomers[END_REF][START_REF] Nagao | Evaluating the Content and Distribution of trans Fatty Acid Isomers in Foods Consumed in Japan[END_REF]. As regards to ruminant meat available at retail (beef and lamb), the presence of TVA is now well-reported (Figure 5B) [START_REF] Guillocheau | Dairy products as main dietary sources of trans-palmitoleic and trans-vaccenic acid in France[END_REF][START_REF] Wolff | Content and distribution of trans-18:1 acids in ruminant milk and meat fats. Their importance in european diets and their effect on human milk[END_REF]. Of note, TVA is sometimes not the major trans-C18:1 isomer in commercial ruminant meat [START_REF] Nagao | Evaluating the Content and Distribution of trans Fatty Acid Isomers in Foods Consumed in Japan[END_REF][START_REF] Aldai | Survey of the fatty acid composition of Canadian beef: Backfat and longissimus lumborum muscle[END_REF][START_REF] Aldai | Can the trans-18:1 and conjugated linoleic acid profiles in retail ground beef be healthier than steak?[END_REF][START_REF] Bravo-Lamas | Characterization of the fatty acid composition of lamb commercially available in northern Spain: Emphasis on the trans-18:1 and CLA content and profile[END_REF], ranging from 10% to 60% of total trans-C18:1 in Canadian stores [START_REF] Aldai | Survey of the fatty acid composition of Canadian beef: Backfat and longissimus lumborum muscle[END_REF]. Consequently, absolute concentrations vary as well, ranging from 0.5% to 3.1% of total fatty acids in Canada [START_REF] Aldai | Survey of the fatty acid composition of Canadian beef: Backfat and longissimus lumborum muscle[END_REF][START_REF] Aldai | Can the trans-18:1 and conjugated linoleic acid profiles in retail ground beef be healthier than steak?[END_REF].

Figure 5 about here.

Because PHOs in general (i.e., PHVOs and PHFOs) are characterised by a wide range of trans-C18:1 positional isomers, these oils do contain TVA. In the late 1990s, Wolff and colleagues reported that TVA accounted for 13.7% of total trans-C18:1 fatty acids in PHVOcontaining foods available in France [START_REF] Wolff | Follow-up of the ∆4 to ∆16 trans-18:1 isomer profile and content in French processed foods containing partially hydrogenated vegetable oils during the period 1995-1999. Analytical and nutritional implications[END_REF]. Lower amounts were reported by Nestlé, who found a mean TVA content of 5.6% of total trans-C18:1 in commercial PHVOs [START_REF] Bezelgues | Formation of trans fatty acids during hydrogenation of edible oils[END_REF]. As for PHFOs, analytical chromatograms showed the presence of TVA but without providing any relative amount [START_REF] Mjøs | Trans fatty acid analyses in samples of marine origin: The risk of false positives[END_REF][START_REF] Kraft | Silver ion solid-phase extraction chromatography for the analysis of trans fatty acids[END_REF].

Therefore, independently of the hypothetic presence of TPA in PHVOs, the TVA present in such oils may in part explain the epidemiological correlation between circulating levels of TPA and intakes of PHVOs [START_REF] Santaren | Serum pentadecanoic acid (15:0), a short-term marker of dairy food intake, is inversely associated with incident type 2 diabetes and its underlying disorders[END_REF][START_REF] Mozaffarian | trans-Palmitoleic acid, other dairy fat biomarkers, and incident diabetes: the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF][START_REF] De Oliveira Otto | Biomarkers of Dairy Fatty Acids and Risk of Cardiovascular Disease in the Multi-Ethnic Study of Atherosclerosis[END_REF][START_REF] Baylin | Adipose tissue biomarkers of fatty acid intake[END_REF] through peroxisomal β-oxidation. Whatever the origin, TVA may indeed undergo peroxisomal β-oxidation and yield TPA. Therefore, both ruminantderived foods and PHOs do contribute to indirect TPA intakes via TVA (Figure 6). Of note, the role of foods which composition is based both on ruminant fat and vegetable fat should be underlined, because they will contribute to dietary TVA intakes. Thus, it is interesting to focus on the relative contribution of ruminant-derived foods and PHVOs to TVA intakes in the general population. To the best of our knowledge, only Wolff and Precht estimated and updated until 2003 these proportions in Europe and the USA based on accurate data on trans-C18:1 profiles in foods [START_REF] Wolff | Follow-up of the ∆4 to ∆16 trans-18:1 isomer profile and content in French processed foods containing partially hydrogenated vegetable oils during the period 1995-1999. Analytical and nutritional implications[END_REF][START_REF] Wolff | Content and distribution of trans-18:1 acids in ruminant milk and meat fats. Their importance in european diets and their effect on human milk[END_REF][START_REF] Wolff | Reassessment of the contribution of bovine milk fats to the trans-18:1 isomeric acid consumption by European populations. Additional data for rumenic (cis-9, trans-11 18:2) acid[END_REF]. For instance, in France and Germany in the late 1990s, ruminantderived foods supplied approx. 80% of the total dietary TVA intakes, the latter being estimated at 900 mg/person/day. Conversely, the relative contribution of ruminant-derived foods in dietary TVA intakes in the USA at the same time was lower than 50%, such intakes being assessed at 1400 mg/person/day.

As said above, it is very likely that PHOs are not used anymore in Western countries, including Western European countries. Should this hypothesis be verified, ruminant-derived foods would, therefore, be the only contributors to TPA intakes in the concerned countries (Figure 7).

Metabolism of TPA in rodents and humans: bridging the gap with other natural trans fatty acids of nutritional interest

Despite the inability for humans to synthesise de novo trans fatty acids, humans are able to metabolise dietary trans fatty acids like any usual fatty acid. Therefore, 2 carbons can be added/removed through the elongation/β-oxidation step, or a cis ethylenic bond can be added in the Δ5, Δ6 or Δ9 position of the chain backbone.

The elongation step of dietary TPA is described in several cell models. In bovine preadipocytes and adipocytes, the addition of TPA in the culture medium led to an increase in intracellular TPA, but also in intracellular TVA [START_REF] Kadegowda | Cis-9,trans-11 conjugated linoleic acid is endogenously synthesized from palmitelaidic (C16:1 trans-9) acid in bovine adipocytes[END_REF]. This elongation step is also reported in endothelial HUVEC cells and hepatic HepG2 cells [START_REF] Da Silva | Trans fatty acids suppress TNFα-induced inflammatory gene expression in endothelial (HUVEC) and hepatocellular carcinoma (HepG2) cells[END_REF]. Our research group confirmed the existence of this elongation step as well in cultured rat hepatocytes: the increase in TPA content in the culture medium was followed by a similar increase in TVA content in hepatocytes (Guillocheau et al, unpublished results). The use of [1-13 C]-TPA in the culture medium allowed the detection of 13 C-labelled TVA, demonstrating that the synthesised TVA did stem from incubated TPA (Guillocheau et al, unpublished results). So far, it remains unknown whether Elongation of very-long chain fatty acids enzyme (ELOVL)-5 or ELOVL-6 is involved in this elongation step.

Conversely, dietary TPA can be chain-shortened. Using cultured rat hepatocytes, we pointed out a 14:1 fatty acid that arose when TPA was added to the culture medium. Relying on [1-13 C]-TPA, the 14:1 fatty acid was not labelled, consistent with a 2 carbon-removal step from TPA (Guillocheau et al, unpublished results). Like TVA being retro-converted to TPA by one cycle of peroxisomal β-oxidation, TPA is also likely to be shortened as well through the same pathway [START_REF] Guillocheau | 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[END_REF].

Apart from elongation and peroxisomal β-oxidation, dietary TPA can theoretically be desaturated. Given that TPA has already a double bond in the Δ9 position, its Δ9-desaturation is not possible. The few studies carried out on cell models did not report any trans-C16:2 fatty acids following the addition of TPA in the culture medium [START_REF] Da Silva | Trans fatty acids suppress TNFα-induced inflammatory gene expression in endothelial (HUVEC) and hepatocellular carcinoma (HepG2) cells[END_REF][START_REF] Kadegowda | Cis-9,trans-11 conjugated linoleic acid is endogenously synthesized from palmitelaidic (C16:1 trans-9) acid in bovine adipocytes[END_REF]. Using cultured rat hepatocytes and increasing amounts of TPA in the culture medium, our research group also failed at highlighting trans-C16:2 fatty acids (Guillocheau et al, unpublished results). Thus, TPA is not likely a suitable substrate for any of the desaturases present in Mammals.

To sum up, dietary TPA undergoes only elongation and peroxisomal β-oxidation reactions in rodents and humans (Figure 8). The elongation step is quite interesting since there is greater knowledge about the metabolism of TVA and its physiological effects. Therefore, it is the opportunity to review the reactions as well that TVA (both dietary and obtained from TPA) undergoes.

Figure 8 about here.

TVA can first be desaturated. It is now well-demonstrated that TVA can be Δ9-desaturated to yield RMA: such a step was pointed out in both rodents [START_REF] Santora | Trans-vaccenic acid is desaturated to conjugated linoleic acid in mice[END_REF][START_REF] Gruffat | Vaccenic acid metabolism in the liver of rat and bovine[END_REF][START_REF] Kraft | The Conversion Efficiency of trans-11 and trans-12 18:1 by Δ9-Desaturation Differs in Rats[END_REF] and humans [START_REF] Turpeinen | Bioconversion of vaccenic acid to conjugated linoleic acid in humans[END_REF][START_REF] Mosley | Cis-9, Trans-11 Conjugated Linoleic Acid Is Synthesized Directly From Vaccenic Acid in Lactating Women[END_REF][START_REF] Kuhnt | Trans-11-18:1 is effectively Δ9-desaturated compared with trans-12-18:1 in humans[END_REF] and would occur with a 20%-conversion efficiency. However, to the best of our knowledge, there is no proof of neither Δ5 nor Δ6-desaturation of TVA even if the reaction could theoretically take place. Of note, however, our research group has recently demonstrated the ability for TVA to undergo a Δ13-desaturation step yielding a conjugated trans-11, cis-13 C18:2 fatty acid. We formally demonstrated the occurrence of such a step in cultured rat hepatocytes [START_REF] Rioux | Trans-vaccenate is Δ13-desaturated by FADS3 in rodents[END_REF], bovine mammary MAC-T and BME-UV endothelial cells [START_REF] Garcia | Synthesis of the suspected trans-11,cis-13 conjugated linoleic acid isomer in ruminant mammary tissue by FADS3-catalyzed Δ13-desaturation of vaccenic acid[END_REF], and in the mammary gland of pregnant rats fed a high-TVA diet [START_REF] Garcia | Conversion of dietary trans-vaccenic acid to trans11,cis13-conjugated linoleic acid in the rat lactating mammary gland by Fatty Acid Desaturase 3-catalyzed methyl-end Δ13desaturation[END_REF]. Apart from desaturation, TVA can be chain-shortened yielding TPA as previously described. Conversely, TVA can be converted to the trans-13 C20:1 fatty acid thanks to an elongation step [START_REF] Rioux | Trans-vaccenate is Δ13-desaturated by FADS3 in rodents[END_REF].

Because RMA has a conjugated double bond system, all the fatty acids that metabolically arise from it will have such a system as well. The main characterized pathway in rodents starting from RMA consists first in a Δ6-desaturation step (cis-6, cis-9, trans-11 C18:3 conjugated fatty acid), second in an elongation step (cis-8, cis-11, trans-13 C20:3 conjugated fatty acid), and third in a probably Δ5-desaturation step (cis-5, cis-8, cis-11, trans-13 C20:4 conjugated fatty acid) [START_REF] Banni | Conjugated linoleic acid metabolism[END_REF][START_REF] Banni | Detection of conjugated C16 PUFAs in rat tissues as possible partial beta-oxidation products of naturally occurring conjugated linoleic acid and its metabolites[END_REF][START_REF] Banni | Conjugated linoleic acids (CLA) as precursors of a distinct family of PUFA[END_REF][START_REF] Sébédio | Metabolites of conjugated isomers of linoleic acid (CLA) in the rat[END_REF][START_REF] Berdeaux | In vitro desaturation and elongation of rumenic acid by rat liver microsomes[END_REF]. Apart from this pathway, RMA is likely to be elongated and converted to the cis-11, trans-13 C20:2 conjugated fatty acid. Of note, several studies pointed out C16:2 and C16:3 conjugated fatty acids probably arising from peroxisomal β-oxidation of RMA and its derivatives [START_REF] Banni | Conjugated linoleic acid metabolism[END_REF][START_REF] Banni | Detection of conjugated C16 PUFAs in rat tissues as possible partial beta-oxidation products of naturally occurring conjugated linoleic acid and its metabolites[END_REF][START_REF] Banni | Conjugated linoleic acids (CLA) as precursors of a distinct family of PUFA[END_REF][START_REF] Sergiel | β-oxidation of conjugated linoleic acid isomers and linoleic acid in rats[END_REF][START_REF] Sébédio | The effect of conjugated linoleic acid isomers on fatty acid profiles of liver and adipose tissues and their conversion to isomers of 16∶2 and 18∶3 conjugated fatty acids in rats[END_REF].

Methodological topics related to TPA

Analysing and quantifying TPA in foods and human tissues

TPA is commercially available as a pure fatty acid methyl ester standard. Thus, relying on gas-chromatography (GC, most of the time coupled with a flame ionization detector, FID) and by comparison of the retention time of a suspected peak, one is, at first sight, able to identify and quantify TPA. For that reason, quantification of TPA is frequently reported in many studies, including epidemiological and food composition ones.

Theoretical considerations about fatty acids analysis by GC are beyond the scope of this review. Still, this review is the occasion to remind how much attention the trans fatty acid analysis requires. Most knowledge is about trans-C18:1 positional isomers, but such knowledge holds true for trans-C16:1 positional isomers as well. Very often, it is believed that TPA is the only trans-C16:1 fatty acid that can be encountered. Instead, when analysing foods [START_REF] Molkentin | Occurrence of trans-C16:1 acids in bovine milkfats and partially hydrogenated edible fats[END_REF], human plasma [START_REF] Jaudszus | trans Palmitoleic acid arises endogenously from dietary vaccenic acid[END_REF] or human milk [START_REF] Precht | Identification and quantitation of cis/trans C16:1 and C17:1 fatty acid positional isomers in German human milk lipids by thin-layer chromatography and gas chromatography/mass spectrometry[END_REF], a wide range of trans-C16:1 positional isomers can be encountered (Figure 4). Resolving all trans-C16:1 positional isomers is therefore mandatory for proper quantification of TPA. For that purpose, both AOCS Ce 1h-05 [START_REF]AOCS, AOCS Official Method Ce 1h-05[END_REF] and AOCS Ce 1j-07 [START_REF]Off. Methods Recomm. Pract. AOCS[END_REF] official methods advocate the use of long and highly polar GC columns: these methods are efficient as regards to the resolution of trans-C18:1 isomers, but they do work as well for trans-C16:1 fatty acids. More precisely, 100 m CP-Sil 88 (Agilent Technologies) [START_REF] Molkentin | Occurrence of trans-C16:1 acids in bovine milkfats and partially hydrogenated edible fats[END_REF][START_REF] Destaillats | Study of individual trans-and cis-16:1 isomers in cow, goat, and ewe cheese fats by gas-liquid chromatography with emphasis on the trans-Δ3 isomer[END_REF], 100 m SP-2560/Rt-2560 (Supelco/Restek) [START_REF] Kramer | Combining results of two GC separations partly achieves determination of all cis and trans 16:1, 18:1, 18:2 and 18:3 except CLA isomers of milk fat as demonstrated using Ag-ion SPE fractionation[END_REF][START_REF] Kraft | Silver ion solid-phase extraction chromatography for the analysis of trans fatty acids[END_REF], 100 m BPX-90 (SGE Analytical Science) (Figure 4) [START_REF] Guillocheau | Dairy products as main dietary sources of trans-palmitoleic and trans-vaccenic acid in France[END_REF][START_REF] Guillocheau | 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[END_REF], 100 m ZB-88 (Phenomenex) [START_REF] Guillocheau | Using GC-MS and helium to resolve positional isomers of trans-C16:1 and trans-C18:1 fatty acids[END_REF] and 100 m SLB-IL 111 (Supelco) [START_REF] Fardin-Kia | Separation of the fatty acids in menhaden oil as methyl esters with a highly polar ionic liquid gas chromatographic column and identification by time of flight mass spectrometry[END_REF][START_REF] Delmonte | Evaluation of highly polar ionic liquid gas chromatographic column for the determination of the fatty acids in milk fat[END_REF][START_REF] Delmonte | Separation characteristics of fatty acid methyl esters using SLB-IL111, a new ionic liquid coated capillary gas chromatographic column[END_REF] GC columns are a strict minimum to resolve trans-C16:1 positional isomers. The 200 m CP-Select FAME (Agilent Technologies) also offers a decent resolution of trans-C16:1 positional isomers [START_REF] Jaudszus | trans Palmitoleic acid arises endogenously from dietary vaccenic acid[END_REF], allowing proper quantification of TPA in foods and plasma.

Furthermore, great attention should be paid to the oven temperature. It is now well demonstrated that isothermal temperature programs are more efficient than gradient temperature programs at resolving trans-C18:1 and trans-C16:1 positional isomers. More precisely, low-temperature isothermal programs should be applied [START_REF] Wolff | Comments on the Resolution of Individual trans-18:1 Isomers by Gas-Liquid Chromatography[END_REF]. Both AOCS official methods recommend a 175 °C isothermal-program [START_REF]AOCS, AOCS Official Method Ce 1h-05[END_REF][START_REF]Off. Methods Recomm. Pract. AOCS[END_REF]. However, this temperature is more suitable for the resolution of trans-C18:1 positional isomers and will not allow a decent resolution of trans-C16:1 positional isomers. In fact, way lower isothermal temperatureprograms (i.e., approx. 125 °C) were successfully used for very nice resolution of trans-C16:1 positional isomers [START_REF] Molkentin | Occurrence of trans-C16:1 acids in bovine milkfats and partially hydrogenated edible fats[END_REF][START_REF] Precht | Identification and quantitation of cis/trans C16:1 and C17:1 fatty acid positional isomers in German human milk lipids by thin-layer chromatography and gas chromatography/mass spectrometry[END_REF][START_REF] Kraft | Silver ion solid-phase extraction chromatography for the analysis of trans fatty acids[END_REF][START_REF] Guillocheau | 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[END_REF][START_REF] Molkentin | Content of individual cis/trans isomers of 16:1, 18:1 and 18:2 fatty acids in the reference milk fat CRM164[END_REF]. Of course, the main drawbacks of using very lowtemperature isothermal programs are the higher retention times of trans-C16:1 fatty acids, which are comprised between 55 and 110 min depending on the carrier gas (H2 and He, respectively).

Above all, dairy fat contains iso-C17:0 fatty acid which overlaps with TPA, even when using highly polar GC columns. The case of iso-C17:0 was pointed out several times as a serious issue when quantifying the so-called "TPA" [START_REF] Destaillats | Study of individual trans-and cis-16:1 isomers in cow, goat, and ewe cheese fats by gas-liquid chromatography with emphasis on the trans-Δ3 isomer[END_REF][START_REF] Precht | Identification and quantitation of cis/trans C16:1 and C17:1 fatty acid positional isomers in German human milk lipids by thin-layer chromatography and gas chromatography/mass spectrometry[END_REF][START_REF] Ratnayake | Concerns about the use of 15:0, 17:0, and trans-16:1n-7 as biomarkers of dairy fat intake in recent observational studies that suggest beneficial effects of dairy food on incidence of diabetes and stroke[END_REF], not only in ruminant-derived foods but also plasma or red blood cells for epidemiological purposes. Furthermore, the widely used flame-ionization detector (FID) cannot distinguish TPA from iso-C17:0, contrary to the mass spectrometer (MS), so that no co-elution can be suspected. To tackle this issue and get reliable quantification of TPA, saturated/cis/trans fractionation based on silver-ion chromatography is mandatory. One can get a fraction containing only trans-MUFA, and perform adequate GC method for analysis. Such a step can be carried out by Ag + -TLC [START_REF] Guillocheau | 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[END_REF][START_REF] Boué | Trans fatty acids in adipose tissue of French women in relation to their dietary sources[END_REF], Ag + -highperformance liquid chromatography (Ag + -HPLC) [START_REF] Delmonte | Evaluation of highly polar ionic liquid gas chromatographic column for the determination of the fatty acids in milk fat[END_REF] or Ag + -solid-phase extraction (Ag + -SPE) [START_REF] Luna | Occurrence of C16:1 isomers in milk fats from ewes fed with different dietary lipid supplements[END_REF][START_REF] Kramer | Combining results of two GC separations partly achieves determination of all cis and trans 16:1, 18:1, 18:2 and 18:3 except CLA isomers of milk fat as demonstrated using Ag-ion SPE fractionation[END_REF][START_REF] Kraft | Silver ion solid-phase extraction chromatography for the analysis of trans fatty acids[END_REF].

Given that TPA may co-elute with other trans-C16:1 positional isomers and/or iso-C17:0, overestimation of TPA content is at risk. Yet, methods to get reliable amounts of TPA are still expensive and burdensome, making them hardly feasible in studies involving many samples or volunteers. Therefore, several compositional studies overestimated the content in TPA in various matrixes. For instance, the so-called "TPA" accounted for 0.8% of total fatty acids in dairy fat according to the TRANSFAIR Study [START_REF] Aro | Trans fatty acids in Dairy and Meat Products from 14 European Countries: the TRANSFAIR Study[END_REF]. This corresponds to a 2,000% overestimation compared with the more reliable data of Destaillats and colleagues who used Ag + -TLC combined with appropriate GC analysis [START_REF] Destaillats | Study of individual trans-and cis-16:1 isomers in cow, goat, and ewe cheese fats by gas-liquid chromatography with emphasis on the trans-Δ3 isomer[END_REF]. As regards to epidemiological studies (Table 1), overestimation is hard to assess given the few available details concerning the methods used. In addition, there is no consensus about what a "normal" plasmatic concentration of TPA in humans is. To our knowledge, only Boué and colleagues applied proper analytical methods on subcutaneous fat of French women to properly assess the content in TPA [START_REF] Boué | Trans fatty acids in adipose tissue of French women in relation to their dietary sources[END_REF]. In that study, TPA accounted for 0.06% of total fatty acids; but this value cannot really be compared to epidemiological studies that were focused on plasma.

Chemical synthesis and isolation of pure TPA for nutritional studies

The exciting outcomes of epidemiological studies about dietary TPA call for in vivo supplementation/nutritional studies (carried out either on rodent or better humans) to unravel hypothetic physiological effects of this fatty acid. Two requests should be met to settle such nutritional studies. On the one hand, one goes beyond actual intakes of TPA and uses high levels of this fatty acid to maximizes chances to highlight probable benefits (i.e., the amount criteria). On the other hand, TPA should be as pure as possible to avoid any bias at drawing conclusions (i.e., the purity criteria).

There is a real challenge which explains why only one in vivo nutritional study focusing on TPA is published so far [START_REF] Cimen | Double bond configuration of palmitoleate is critical for atheroprotection[END_REF]. On the one hand, usual chemical suppliers (e.g., Sigma-Aldrich) do offer a > 98%-purity TPA but only several hundreds of mg are available. Besides, food sources of TPA meet neither the purity criteria (e.g., presence of other fatty acids that require to be removed) nor the amount criteria. Of note, however, the Palmitoleic Isomer Study relies on PHVOs for the clinical supplementation in TPA, suggesting an ability to remove the other trans-C16:1 positional isomers that are contained in PHVOs [START_REF] Lichtenstein | Palmitoleic Isomer Study[END_REF].

Chemical synthesis is maybe the best strategy to get high amounts of pure TPA. Despite no published method to synthesize TPA so far, one can rely on the couple of studies that investigated the physiological effects of TVA and relied on chemical synthesis. Because TVA is a trans-MUFA, any methodology used for chemical synthesis of TVA could be translated to the case of TPA. The typical workflow consists of a Wittig reaction for the synthesis of the corresponding cis-MUFA, then followed by an isomerisation and afterwards by a large-scale cis/trans fractionation [START_REF] Guillocheau | 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[END_REF][START_REF] Garcia | Conversion of dietary trans-vaccenic acid to trans11,cis13-conjugated linoleic acid in the rat lactating mammary gland by Fatty Acid Desaturase 3-catalyzed methyl-end Δ13desaturation[END_REF][START_REF] Fusari | Syntheses of Cis-and Trans-7-and 8-Octadecenoic Acids : Comparison of the Properties of Cis-and Trans-6-, 7-, 8-, 9-, and 11-Octadecenoic Acids[END_REF][START_REF] Mouloungui | Chemical synthesis of monounsaturated trans fatty acids[END_REF][START_REF] Tyburczy | Individual Trans Octadecenoic Acids and Partially Hydrogenated Vegetable Oil Differentially Affect Hepatic Lipid and Lipoprotein Metabolism in Golden Syrian Hamsters[END_REF][START_REF] Wang | The intestinal bioavailability of vaccenic acid and activation of peroxisome proliferator-activated receptor-α and -γ in a rodent model of dyslipidemia and the metabolic syndrome[END_REF][START_REF] Mohankumar | Dietary supplementation of trans-11-vaccenic acid reduces adipocyte size but neither aggravates nor attenuates obesity-mediated metabolic abnormalities in fa/fa Zucker rats[END_REF][START_REF] Gebauer | Effect of trans fatty acid isomers from ruminant sources on risk factors of cardiovascular disease: study design and rationale[END_REF][START_REF] Gebauer | Vaccenic acid and trans fatty acid isomers from partially hydrogenated oil both adversely affect LDL cholesterol: a double-blind, randomized controlled trial[END_REF][START_REF] Duffy | Synthesis of trans-vaccenic acid and cis-9-trans-11-conjugated linoleic acid[END_REF][START_REF] Ahmad | Synthesis of cis-11-Octadecenoic and trans-11-Octadecenoic (Vaccenic) Acids[END_REF]. As regards to the purity criteria, the Wittig reaction is strategic: the position of the double bond is certain, thus avoiding any other positional isomer of the cis-MUFA [START_REF] Mouloungui | Chemical synthesis of monounsaturated trans fatty acids[END_REF][START_REF] Duffy | Synthesis of trans-vaccenic acid and cis-9-trans-11-conjugated linoleic acid[END_REF]. The subsequent isomerisation reaction is nowadays well described and yields a mix of trans/cis MUFA (approx. 80/20) [START_REF] Mouloungui | Chemical synthesis of monounsaturated trans fatty acids[END_REF][START_REF] Duffy | Synthesis of trans-vaccenic acid and cis-9-trans-11-conjugated linoleic acid[END_REF]. Large scale cis/trans purification to remove the cis-MUFA is usually achieved by means of low-temperature solvent crystallization [START_REF] Fusari | Syntheses of Cis-and Trans-7-and 8-Octadecenoic Acids : Comparison of the Properties of Cis-and Trans-6-, 7-, 8-, 9-, and 11-Octadecenoic Acids[END_REF][START_REF] Mouloungui | Chemical synthesis of monounsaturated trans fatty acids[END_REF][START_REF] Gebauer | Effect of trans fatty acid isomers from ruminant sources on risk factors of cardiovascular disease: study design and rationale[END_REF][START_REF] Duffy | Synthesis of trans-vaccenic acid and cis-9-trans-11-conjugated linoleic acid[END_REF][START_REF] Ahmad | Synthesis of cis-11-Octadecenoic and trans-11-Octadecenoic (Vaccenic) Acids[END_REF]: acetone and ether are widely used, but methanol is also a suitable solvent for that purpose. Alternatively, flash liquid-chromatography (LC) was used by our research group for successful large-scale cis/trans fractionation of TVA. In all case, purities greater than 95% were achieved for TVA [START_REF] Guillocheau | 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[END_REF][START_REF] Garcia | Conversion of dietary trans-vaccenic acid to trans11,cis13-conjugated linoleic acid in the rat lactating mammary gland by Fatty Acid Desaturase 3-catalyzed methyl-end Δ13desaturation[END_REF].

Otherwise, and specifically to the case of TPA, one can also use food sources of CPA instead of relying on Wittig reaction. CPA is contained in elevated amounts in macadamia nut oil (approx. 15% of total fatty acids) or better in the dietary complement Provinal ® (> 50% of total fatty acids). Importantly, these sources have very low amounts of other C16:1 positional isomers. Flash-LC can be used for purification of CPA, and the previously described steps for getting pure TPA can be subsequently applied [START_REF] Guillocheau | Supplementation studies involving natural trans fatty acids: real technical challenges, actual solutions[END_REF].

Conclusion and perspectives

To our knowledge, this is the very first review specifically dedicated to TPA. We discussed here a very wide range of aspects related to TPA: suggested nutritional benefits, origin, occurrence in foods and metabolism. We also developed on methodological topics related to this fatty acid, which are quite important for future epidemiological and supplementation studies. Importantly, we reviewed articles dealing specifically with TPA, to avoid any confusion with its cis counterpart.

We began our review reminding the biochemical definition of TPA, that is the location and the configuration of its double bond. Both nomenclatures (Ω and Δ) were used to makes things quite clear. Based on the wide range of denominations that can be found in the literature, it is very easy for non-specialists to be confused: "palmitoleic acid", "palmitelaidic acid", "C16:1", "trans-C16:1", "C16:1 n7", "tC16:1 n-9"… We believe it is mandatory for future research dealing with TPA to accurately state the biochemical formula of the given fatty acid and the nomenclature used, to avoid any misunderstanding.

Because TPA is a trans fatty acid, methodological aspects are of great importance. This review debunked the too widespread idea according to which quantifying TPA in foods or human plasma can be easily performed. Our research group is now experienced with quantification of TPA in foods, and this review is the opportunity to say that quantifying TPA has nothing to do with the measurement of other well-known fatty acids. For instance, proper assessment of circulating levels of EPA and DHA can be easily performed by direct GC, without any burdensome steps. Instead, specific and crucial steps are mandatory for proper quantification of TPA. Thus, future research focusing on TPA should be very accurate on this point as well, detailing the methods performed and the operating conditions if GC is used. Furthermore, we reviewed the methods allowing chemical synthesis and purification of TPA, making supplementation studies on rodents or even humans feasible.

To sum up, TPA is an R-TFA which intakes are exclusively ensured by ruminant-derived foods (i.e., ruminant milk and ruminant meat). Several epidemiological studies (cross-sectional and prospective) suggested positive physiological impacts of dietary TPA with respect to the risk of type 2 diabetes. Importantly, they indicated that dietary TPA could prevent insulinresistance by targeting insulinemia. In vitro studies provided complementary results and showed that TPA could favourably target pancreas and modulate β-cell function. Therefore, there is a call for further research and especially TPA-supplementation studies focused on type 2 diabetes, to unravel mechanisms of action. Furthermore, one cannot exclude that dietary TPA targets other tissues (e.g., liver, adipose tissue).

The metabolism of TPA is a crucial feature. Indeed, it reveals that the whole topic of TPA is strongly tied to that of TVA and RMA. Of note, TVA and RMA have been extensively explored for their health benefits. Thus, it is striking that epidemiological studies focusing on TPA did not make a hint to the metabolism of TPA. Indeed, supplementation studies on rodents dealing with TVA and RMA also pointed out the ability to prevent from insulin-resistance and type 2 diabetes: such results strongly concur with epidemiological outcomes. Therefore, epidemiological studies are totally complementary to supplementation studies carried out on rodents, giving for the first time evidence of benefits of R-TFAs in humans. Beyond, one can wonder if the shared trans n-7 double bond confers special properties to these fatty acids. It is worth underlining that TVA and RMA have been proposed as powerful ligands of PPAR-α and PPAR-γ to explain their benefits in rodents [START_REF] Wang | The intestinal bioavailability of vaccenic acid and activation of peroxisome proliferator-activated receptor-α and -γ in a rodent model of dyslipidemia and the metabolic syndrome[END_REF][START_REF] Moya-Camarena | Conjugated linoleic acid is a potent naturally occurring ligand and activator of PPARα[END_REF]. Can dietary TPA act the same way? Awaiting for well-designed studies, we can reasonably assume that TPA, TVA and RMA are ligands of PPAR-α and PPAR-γ and can act independently of each other.

Finally, the very first question on TPA was about the closeness with CPA, with respect to physiological impacts [START_REF] Mozaffarian | Trans-palmitoleic acid, metabolic risk factors, and new-onset diabetes in U.S. adults[END_REF]. Can a cis-MUFA and a trans-MUFA exert the same physiological effects? Once again, the relationship between fatty acid structure (double bond configuration) and function is crucial. To our knowledge, similar functions between a cis-MUFA and a trans-MUFA have never been proven, making such assumption very intriguing. Thus, supplementation studies on rodents or humans are needed.

TABLE 1

Epidemiological associations between circulating trans-palmitoleic acid in humans and risk of type 2 diabetes, risk of cardiovascular diseases, risk of stroke, anthropometric parameters and metabolic disorders.

Structure of trans-palmitoleic acid: the position of the trans double bond is indicated using either (A) the Δ-nomenclature or (B) the Ω-nomenclature.

FIGURE 2

Origin of trans-palmitoleic acid in ruminant-derived foods: from ruminal biohydrogenation to uptake and metabolism in key ruminant tissues. Hypotheses are illustrated by dotted arrows.

Abbreviations. ALA, α-linolenic acid; LA, linoleic acid; RMA, rumenic acid; TPA, transpalmitoleic acid; TVA, trans-vaccenic acid.

Symbols. β-ox, peroxisomal β-oxidation; Δ, desaturation ε, elongation.

FIGURE 3

Generation of trans-palmitoleic acid from ruminal biohydrogenation of dietary C16:3 n-3, similarly to ruminal biohydrogenation of dietary α-linolenic acid. Red-labelled double bonds undergo subsequent isomerization or saturation reaction.

Abbreviations. ALA, α-linolenic acid; TPA, trans-palmitoleic acid; TVA, trans-vaccenic acid.

FIGURE 4

Typical chromatogram of trans-C16:1 isomers in ruminant-derived foods available at retail in France in 2018, to accurately quantify trans-palmitoleic acid. A: ruminant milk. B: ruminant meat. Fatty acids were analysed by Ag + -TLC followed by GC-MS analysis carried out with the BPX-90 column. Data from [START_REF] Guillocheau | Dairy products as main dietary sources of trans-palmitoleic and trans-vaccenic acid in France[END_REF].

Abbreviations. TPA, trans-palmitoleic acid.

FIGURE 5

Typical chromatogram of trans-C18:1 isomers in ruminant-derived foods available at retail in France in 2018, to accurately quantify trans-vaccenic acid. A: ruminant milk. B: ruminant meat. Fatty acids were analysed by Ag + -TLC followed by GC-MS analysis carried out with the BPX-90 column. Data from [START_REF] Guillocheau | Dairy products as main dietary sources of trans-palmitoleic and trans-vaccenic acid in France[END_REF].

Abbreviations. TVA, trans-vaccenic acid.

FIGURE 6

Dietary sources of trans-palmitoleic acid at the time of the use of partially hydrogenated oils.

Abbreviations. TPA, trans-palmitoleic acid; TVA, trans-vaccenic acid.

FIGURE 7

Dietary sources of trans-palmitoleic acid after regulation measures on partially hydrogenated oils.

Abbreviations. PHFO, partially hydrogenated fish oil; PHO, partially hydrogenated oil; PHVO, partially hydrogenated vegetable oil; TPA, trans-palmitoleic acid; TVA, trans-vaccenic acid.

FIGURE 8

Metabolism of dietary trans-palmitoleic acid in humans and rodents.

Abbreviations. TPA, trans-palmitoleic acid; TVA, trans-vaccenic acid.

Symbols. β-ox, peroxisomal β-oxidation; Δ, desaturation ε, elongation.

FIGURE 1

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B A

Figure 1

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Figure 2

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Figure 3

 3 Figure 3 about here.When it comes to human nutrition, ruminant meat and milk are of high interest. TPA generated through ruminal biohydrogenation may enter the general circulation of the ruminant and be incorporated in muscle and mammary gland. Because fatty acids including trans ones can be metabolised, another question arises about chain-shortening of TVA: does peroxisomal

Figure 4

 4 Figure 4 about here.

Figure 6

 6 Figure 6 about here.

Figure 7

 7 Figure 7 about here.

TABLE 1

 1 Continued. BMI, body mass index; CHS, Cardiovascular Health Study; CRP, C-reactive protein; EPIC, European Prospective Investigation into Cancer and Nutrition; GC-FID, gas chromatography coupled with flame-ionization detector; GC-MS, gas-chromatography coupled with mass spectrometry; HOMA-IR, Homeostasis Model Assessment of Insulin Resistance; HPFS, Health Professionals Follow-up Study; HR, hazard ratio; IFN-γ, Interferon-γ; IL, Interleukin; IRAS, Insulin Resistance Atherosclerosis Study; MESA, Multi-Ethnic Study of Atherosclerosis; NAFLD, nonalcoholic fatty liver disease; NHS, Nurses' Health Study; OR, odds ratio; PL, phospholipids; Q, quartile/quintile (depending on studies); RBC, red blood cells; RR, relative risk; TAG, triacylglycerol.Symbols. ✖, non-significant impact; significant increase; significant decrease.

	Reference	Type of study	Country	Year at blood sampling	Participants n	Human tissue analysed	Circulating TPA Assessment of TPA 1	TPA content 2	Outcomes
							GC-FID
	[19]	Case-control (healthy/obese)	Canada	2004-2007	200	Plasma (PL)	HP-88 100 m × 0.25 mm × 0.20 µm He	Not reported	TPA ⇒ TPA ⇒	BMI Insulinemia
	[11,25]	Prospective EPIC-Potsdam Study	Germany	1994-1998	2724	RBC (PL)	GC-FID 185-245 °C, gradient, N2 CP-Select 100 m × 0.25 mm × 0.20 µm	Q1: 0.12% 3 Q4: 0.27% 3	✖ RR Type 2 diabetes
									HR Type 2 diabetes
									✖ BMI
									Waist girth
									Fasting plasma TAG
									✖ Fasting glycemia
							GC-FID		Fasting insulinemia
	[8]	Prospective CHS Study	USA	1989-1993	3736	Plasma (PL)	SP-2560 100 m × 0.25 mm × 0.20 µm 160-240 °C, gradient	Q1: 0.13% 3 Q4: 0.25% 3	HOMA-IR CRP ✖ LDL-cholesterol
									HDL-cholesterol
									Total cholesterol
									HDL-cholesterol ✖ Diastolic blood pressure
									✖ Systolic blood pressure
	[12]	Prospective EPIC-Norfolk Study	UK	1993-1997	383	Plasma (PL) (PL) RBC	GC-FID	Plasma 0.18% 3 0.06% 3 RBC	✖ OR Type 2 diabetes

TABLE 2

 2 Epidemiological associations between dietary intakes and circulating levels of trans-palmitoleic acid in humans.

	Reference	Type of study	Country	Year at blood sampling	Participants n	Dietary TPA Assessment of dietary intake Food composition data	Human tissue analysed	Circulating TPA Assessment of TPA 1	TPA content 2	Conclusions Dietary sources of TPA
									GC-FID
	[56]	Cross-sectional	Costa-Rica	1994-1998	503	FFQ	USDA database	Subcutaneous adipose tissue	SP-2560 100 m × 0.25 mm × 0.20 µm	0.08% 3	Vegetable fat
									90-240 °C, gradient, H2
							Australian
	[33]	Cross-sectional	Australia	Not reported	86	4-day record	Food Composition	Plasma (PL)	GC-MS BPX-70	Not reported	Dairy products
							tables
									GC-FID
	[24]	Cross-sectional	Canada	> 2009	233	FFQ	NDSR Software	Plasma (PL)	CP-Select 100 m × 0.25 mm × 0.20 µm	0.16% 3	Dairy products
									185-245 °C, gradient
									GC-FID
	[34,91]	Cross-sectional	Netherlands	2006-2013	769 [34] 864 [91]	FFQ	Dutch food database	Plasma (PL)	CP-Select 200 m × 0.25 mm × 0.20 µm	0.020% 4	Dairy products
									190-240 °C, gradient
								Plasma	GC-FID	Plasma
	[35]	Prospective NHS Study	USA	1989-1990	313	FFQ	USDA database	(total lipids) RBC	SP-2560 100 m × 0.25 mm × 0.20 µm	0.15% 3 RBC	Dairy products
								(total lipids)	90-250 °C, gradient, H2	0.14% 3
	[8,36]	Prospective CHS Study	USA	1989-1993	3330	FFQ	Harvard University nutrition database	Plasma (PL)	GC-FID SP-2560 100 m × 0.25 mm × 0.20 µm 160-240 °C, gradient	Not reported	Dairy products Red meat
									GC-FID		Dairy
	[15,22]	Prospective MESA Study	USA	2000-2002	2617 [15] 2837 [22]	FFQ	Not reported	Plasma (PL)	CP-Select 100 m × 0.25 mm × 0.20 µm	0.05% 3	products Red meat
									190-240 °C, gradient		PHVOs
	[13,141]	Prospective IRAS Study	USA	1992-1994	659	FFQ	HHHQ-DIETSYS Software	Serum (total lipids)	GC-FID and GC-MS 30 m × 0.25 mm × 0.20 µm HP-88	0.30% 3	PHVOs

Details about the method for assessment of TPA content are given as follows (if reported in the corresponding study): type of chromatographytype of detector/type of GC column/length of the GC column × inner diameter of the GC column × film thickness of the GC column/startingending temperature of the oven, type of temperature program (gradient or isothermal), carrier

gas.[START_REF] Efsa | Opinion of the Scientific Panel on Dietetic Products , Nutrition and Allergies on a request from the Commission related to the presence of trans fatty acids in foods and the effect on human health of the consumption of trans fatty acids[END_REF] Mean TPA content (if reported in the corresponding study) of either the total subject sample or of the quartiles/quintiles of the corresponding

study.[START_REF][END_REF] Mean TPA content expressed as % of total

fatty acids.[START_REF] Cao | Identification of a Lipokine, a Lipid Hormone Linking Adipose Tissue to Systemic Metabolism[END_REF] Mean TPA content expressed as % mol of total fatty acids.

Acknowledgements

EG acknowledges a CIFRE (Industrial Agreement of Training through Research) PhD fellowship from both the French Dairy Interbranch Organization (CNIEL) and the National Association for Research and Technology (ANRT) (CIFRE fellowship number 2015/1195).

layer chromatography TPA: trans-palmitoleic acid TVA: trans-vaccenic acid

Funding

The present research was financially supported by the French Dairy Interbranch Organization (CNIEL) (PALMITO project) and the Lipids and Nutrition Group (GLN).

Conflicts of interest

EG is employed by the French Dairy Interbranch Organization (CNIEL).

Author contributions

All authors have equally contributed to the review. Dairy products 1 Details about the method for assessment of TPA content are given as follows (if reported in the corresponding study): type of chromatographytype of detector/type of GC column/length of the GC column × inner diameter of the GC column × film thickness of the GC column/startingending temperature of the oven, type of temperature program (gradient or isothermal), carrier gas. 2 Mean TPA content (if reported in the corresponding study) of either the total subject sample or of the quartiles of the corresponding study. 3 Mean TPA content expressed as % of total fatty acids. 4 Mean TPA content expressed as % mol of total fatty acids.

Abbreviations