Hexadecenoic Fatty Acid Isomers: A Chemical Biology Approach for

Article pubs.acs.org/crt

Hexadecenoic Fatty Acid Isomers: A Chemical Biology Approach for Human Plasma Biomarker Development Anna Sansone, Michele Melchiorre, Chryssostomos Chatgilialoglu, and Carla Ferreri* ISOF, Consiglio Nazionale delle Ricerche, Via P. Gobetti 101, 40129 Bologna, Italy S Supporting Information *

ABSTRACT: Hexadecenoic fatty acids are monounsaturated lipid components, which are interesting targets of plasma lipidomic studies and biomarker development. The main positional isomers, palmitoleic (9-cis-16:1) and sapienic acids (6-cis-16:1), have an endogenous origin from palmitic acid, the former being recognized as a component of adipose tissue with signaling activity, whereas the latter is mainly reported as a component of sebum. The trans 16:1 isomers are attributed so far to dietary sources of industrial and dairy fats, whereas the endogenous formation due to the free radical-mediated isomerization can represent an emerging, yet unexplored, pathway connected to cellular stress. Herein, we report a chemical biology approach for the development of hexadecenoic fatty acids as plasma biomarkers, with the first synthesis of 6trans-16:1 and the efficient analytical setup with unambiguous assignment of 16:1 double bond position and geometry, which was applied to human commercial LDL and plasma cholesteryl esters. Sapienic acid was identified together with its geometrical trans isomer for the first time. The quantitation of hexadecenoic fatty acid isomers evidenced their different levels in the two lipid classes and LDL fractions, making us foresee interesting applications to the metabolic evaluation of fatty acid pathways. These findings open new perspectives for plasma lipidomics involving monounsaturated fatty acids, highlighting future developments for their evaluation in different health conditions including free radical stress.

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INTRODUCTION Monounsaturated fatty acid (MUFA) residues are ubiquitously present in the lipids of all living organisms, obtained biosynthetically by desaturase enzymes in a regio- and stereospecific manner. The double bond is created exclusively in the cis configuration, the trans configuration being not synthesized in mammals. As a matter of fact, trans fatty acids in mammals can be due to two main contributions: (i) cellular stress conditions with the formation of free radicals, which have been found to act as effective catalysts of the cis−trans double bond isomerization;1,2 and (ii) the “classical” origin by dairy or industrial fats, raising concern on the health effects such as cholesterol and LDL increase as well as other metabolic and infant growth problems.3−7 It is worth noting that the geometrical trans MUFA isomers were carefully analyzed and studied as products of the enzymatic isomerization process, which is part of the bacterial adaptive response to environmental and temperature stress.8,9 Among MUFA, the hexadecenoic fatty acid family has been recently highlighted for metabolic pathways related to human health conditions. The main representative is palmitoleic acid (9-cis-16:1) derived from palmitic acid (16:0) by the enzymatic activity of delta-9 desaturase enzyme (stearoyl-CoA desaturase, SCD1), with very scarce interference from dietary sources (Figure 1).10 This pathway and the corresponding palmitoleic acid level have been evoked in the etiology of several diseases, © 2013 American Chemical Society

Figure 1. Main biosynthetic pathways from palmitic acid and linoleic acid.

such as obesity, diabetes, and cancer. Plasma phospholipids of homozygote twins were studied by mass spectrometry evidencing that in the obese twin there is an increased level of 9-cis-16:1 compared to that in the nonobese twin.11 In several other reports on obesity and metabolic diseases, this fatty acid was highlighted as an important lipid biomarker of the enzymatic transformation of palmitic acid. In fact, it was defined as an adipose tissue-derived lipid hormone,12 and from the first Received: August 2, 2013 Published: October 2, 2013 1703

dx.doi.org/10.1021/tx400287u | Chem. Res. Toxicol. 2013, 26, 1703−1709

Chemical Research in Toxicology

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Synthesis of 6-trans-16:1 Methyl Ester. A 15 mM solution of sapienic acid methyl ester (4 mg, 0.015 mmol) in 2-propanol (1 mL) was transferred to a quartz photochemical reactor, and the solution was flushed with argon for 20 min to evacuate oxygen. Then, 25 μL of a 0.28 M solution of 2-mercaptoethanol in 2-propanol (previously flushed with argon) was added to the reactor (0.007 mmol), and UV photoirradiation with a 5.5 W low pressure mercury lamp was carried out at (22 ± 2) °C for 4 min.36 The solvent was removed by a rotary evaporator, the residue was dissolved in n-hexane and analyzed by GC revealing a mixture of cis− trans isomers (3.9 mg, with an 8:2 6-trans:6-cis ratio) as is shown in Figure 2 (see also Figure S2 in Supporting Information). One portion

observation of stimulating activity on muscle insulin action in a genetic mouse model, experimental evidence was reported also in humans.13−17 Palmitoleic acid was also found to be involved in cell growth signaling,18 and indeed cell proliferation requiring the de novo lipid biosynthesis can be monitored at early stages by an increase of endogenous MUFA production, as it has been proposed by carcinogenesis studies.19 In the hexadecenoic fatty acid series, a positional isomer having the double bond in the C6 position (6-cis-16:1) known as sapienic acid has been described in sebaceous glands during the differentiation of sebocytes,20−23 in the epidermal lipid surface24,25 and in diseases such as acne,26 atopic dermatitis,27 and rosacea.28 The antimicrobial activity of this unusual fatty acid was evidenced in other studies29,30 and brought as motif of its presence at the epidermal level. Its biosynthesis starts from palmitic acid and involves the delta-6 desaturase (fatty acid desaturase, FADS) enzymes. Figure 1 summarizes the enzymatic processes on palmitic acid giving palmitoleic and sapienic acids, which are the main biosynthetic products of the hexadecenoic fatty acid family and can be found in biological samples. It is worth noting that delta6 desaturase is normally involved in PUFA biosynthesis (in Figure 1, as an example, the omega-6 transformation is shown in blue; the parallel involvement of delta-6 desaturase in the omega-3 pathway is not shown), and its diversion for processing saturated fatty acids (Figure 1, pathway in red) has an interesting, yet fully unexplored, meaning. The presence of hexadecenoic fatty acids in human and animal milk is known;31,32 however, no data are available for the presence of sapienic acid in body compartments other than skin-related tissues. Therefore, its presence in plasma is unknown. The corresponding trans isomers, namely, palmitelaidic acid (9-trans-16:1) and trans-sapienic acid (6-trans-16:1), are attributed to diets rich in manipulated fats by partial hydrogenation, as well as to the consumption of milk products where the trans fats derive from biohydrogenation processes occurring in ruminants. 32 Palmitelaidic acid was also individuated in plasma phospholipids of a cohort of 3736 adults, attributing again its presence to the consumption of dairy products.33 It is worth noting that palmitelaidic acid can be recognized by using the commercially available reference, whereas for trans-sapienic acid, there is no original reference, and in all reports, the proper identification is lacking. In the frame of our chemical biology approach based on the cis and trans lipid library for biomarker development of metabolic and radical stress,34−36 we were interested in expanding the knowledge on the main components of the hexadecenoic fatty acid family. Herein, we wish to report (i) the synthesis and characterization of the geometrical trans isomer of sapienic acid and the development of a satisfactory protocol for separation and unambiguous determination of the main four hexadecenoic MUFA isomers; and (ii) the application of this protocol to the detection and quantitation of these isomers evidencing their different levels in lipid classes of human LDL and plasma cholesteryl esters, thus foreseeing an application for the evaluation of fatty acid pathways and metabolic conditions. These data can contribute to the wide scenario of plasma lipidomics and related biomarkers, with the role of the natural cis geometry as well as the effect of radical stress that transforms it into the trans isomer.

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Figure 2. Representative GC regions corresponding to the main isomers of hexadecenoic fatty acid methyl esters in human plasma CE and LDL fractions. The minor components are individuated for comparison with references, when available. Trace I: standard references of methyl esters (A) 6-trans-16:1, (B) 9-trans-16:1, (C) 6-cis-16:1, and (D) 9-cis-16:1. Trace II: cholesteryl ester (CE) fraction isolated from human plasma. Trace III: triglyceride (TG) fraction of human LDL. Trace IV: CE fraction of human LDL. Trace V: phospholipid (PL) fraction of human LDL. Trace VI: total lipids of human LDL.

(3 mg) of this crude reaction was also chromatographed on an AgTLC plate using 70:30 n-hexane/diethyl ether as the eluent; the area corresponding to the 6-trans isomer was scraped off, and silica gel was washed with 3 mL of chloroform. The purity of the compound was checked by Ag/TLC using the above specified eluent (Rf = 0.65) (Figure S1, Supporting Information).37 The solvent was evaporated, and the pure trans isomer (2.2 mg, 73% yield) was obtained as a transparent oil and fully characterized. 1H NMR (CDCl3) δ 0.89 (t, J = 4 Hz, 3H); 1.26−1.39 (m, 14H); 1.54−1.66 (m, 4H); 1.94−2.01 (m, 4H); 2.32 (t, J = 8 Hz, 2H); 3.67 (s, 3H, OCH3); 5.32−5.44 (m, 2H). 13 C NMR δ 14.3, 22.9, 24.7, 29.3, 29.4, 29.6, 29.7, 29.8, 29.9, 32.1, 32.4, 32.8, 34.2, 51.3, 129.7, 131.2, 174.3. IR (CHCl3) cm−1 3015 (ν CH); 2956 (ν CH2); 2927 (ν CH2); 1738 (ν COO), 1420 (ν CH2), 972 (ν =CH); GC/MS (C17H32O2; mw 268).

EXPERIMENTAL PROCEDURES

Materials and Methods. See Supporting Information. 1704



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Table 1. Hexadecenoic Fatty Acid Composition (% rel), Following the Elution Order in the GC Analysis of the Corresponding Fatty Acid Methyl Esters, Found in Human Plasma Cholesteryl Esters (CE) (n = 5) and Commercial Human LDL Samples (n = 5), As Total Lipids and the Fractions of Triglycerides (TG), Cholesteryl Esters (CE), and Phospholipids (PL) FAMEa 6-trans 9-trans 6-cis 9-cis

total LDL lipids

CE LDL

± ± ± ±

0.15 ± 0.02 nd 0.79 ± 0.07 2.10 ± 0.67

0.10 0.01 1.32 1.60

0.01 0.01 0.40 0.40

PL LDL

TG LDL

human plasma CE

± ± ± ±

nd nd 5.45 ± 0.86 1.19 ± 0.43

0.05 ± 0.01 nd 0.94 ± 0.23 1.74 ± 0.44

0.05 0.02 0.31 0.42

0.01 0.01 0.08 0.03

FAME expressed as % rel of the total fatty acid peak areas detected in the GC chromatogram and reported as the mean ± sd of the experiments as indicated in the legend (see Supporting Information for the full composition); nd = not detectable.

a

Lipid Analysis of Human LDL. Samples of commercial LDL from human plasma were treated by two different protocols (see Scheme S2, Supporting Information): Protocol 1. To an LDL sample (2 mg), brine was added (4 mL), and the sample was extracted with 2:1 chloroform/methanol (4 × 4 mL). The organic layers were dried on anhydrous Na2SO4 and evaporated to dryness. TLC revealed that the crude was composed of triglycerides, phospholipids, cholesteryl esters, and cholesterol as described in the literature.38 This mixture was added with 0.5 mL of 1 M solution of NaOH in 2:3 benzene/methanol and stirred in the dark under argon for 25 min at room temperature. The reaction was quenched with brine (0.5 mL) and extracted with n-hexane (3 × 2 mL) to afford a crude containing fatty acid methyl esters, which were analyzed by GC (see details in Supporting Information). The procedure was applied to a total number of 5 samples of commercial LDL. Protocol 2. A commercial sample of LDL (2 mg) was extracted as described above. The extraction was followed by the separation of lipid classes by thin-layer chromatography with hexane/diethylether/acetic acid (70:30:1) as eluent; cholesteryl esters, triglycerides, and phospholipids were obtained and converted to the corresponding FAME by appropriate methods, as follows: the cholesteryl ester fraction was treated by adding 0.2 mL of a 1 M solution of NaOH in 2:3 benzene/methanol and stirring in the dark under argon for 15 min at room temperature, followed by quenching with brine. The workup was the same as that indicated in protocol 1, and the reaction crude containing FAME was examined by GC. The triglyceride fraction was treated by adding 0.2 mL of a 1 M solution of NaOH in 2:3 benzene/ methanol and stirring in the dark under argon for 3 min at room temperature, followed by quenching with brine. The workup was the same as that indicated for cholesteryl esters. The phospholipid fraction was treated by adding 0.2 mL of a 0.5 M solution of KOH in methanol, stirring the reaction under argon for 5 min at room temperature; after quenching with brine, the workup was the same as that indicated for cholesteryl esters and triglycerides. The procedure was applied to a total number of 5 samples of commercial LDL (see details in Supporting Information). Analysis of Cholesteryl Esters from Human Plasma. Cholesteryl esters from human plasma were obtained following previously reported procedures.36 From samples of human blood plasma (0.5 mL), a total lipid crude fraction (ca. 7−9 mg) was dissolved in n-hexane (1 mL) and loaded on a silica gel cartridge (1g silica gel) conditioned with n-hexane. Using 9:1 n-hexane/diethylether as the eluent, the fraction containing cholesteryl esters was isolated, collected (ca. 1.5 mg), and converted to the fatty acid methyl esters as described above (see details in Supporting Information). The procedure was applied to a total number of 5 samples of human plasma. DMDS Derivatization of Fatty Acid Methyl Esters. Sapienic acid methyl ester was treated following the literature procedure.39,40 Briefly, a solution of sapienic acid methyl ester in n-hexane (50 μL of a 1.8 mM solution) in a Wheaton vial was added with 100 μL of dimethyl disulfide and 2 drops of a 6% solution of iodine in diethyl ether. The reaction was stirred at 38 °C for 36 h, then 1 mL of nhexane and 1 mL of a 5% aqueous solution of sodium thiosulphate were consecutively added. The organic phase was isolated, dried over anhydrous Na2SO4, and concentrated under a gentle stream of

nitrogen before the GC-MS analysis. The FAME extracts from LDL and cholesteryl esters were treated following the procedure described above.

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RESULTS Synthesis and Characterization of 6-trans-Hexadecenoic Acid Methyl Ester. The photoisomerization reaction represents a flexible methodology to prepare selectively geometrical trans isomers since no positional shift of the double bond occurs. 1,2 A degassed solution of 6-cishexadecenoic acid methyl ester in 2-propanol was irradiated in the presence of 50% molar equivalents of 2-mercaptoethanol (Scheme S1, Supporting Information). The reaction course was monitored by silver-thin layer chromatography (Ag-TLC),37 which was also used to isolate the trans isomer fraction in a 73% yield, which turned out to be a quantitative yield after the recovery of the unreacted cis isomer (Figure S1, Supporting Information). The new compound was fully characterized (see Supporting Information). 1H and 13C NMR spectra showed resonance shifts distinguishable from the corresponding cis isomer, as already described for other geometrical isomers (Figure S3, Supporting Information).34,36 In particular, the 13C NMR spectrum showed the ethylenic carbon atom resonances of the trans isomer at 129.7 and 131.2 ppm, for the C6 and C7 carbon atoms, whereas the cis isomer has the C6 and C7 resonances at 129.3 and 130.7 ppm, respectively. The IR methodology, which is a well known tool for cis and trans lipid analysis, confirmed the presence of the characteristic band due to the deformation of the C−H bond adjacent to the trans double bond at 972 cm−1 (Figure S4, Supporting Information).41 GC Analysis. Having four isomers of hexadecenoic acid methyl esters in our hands, we set up a gas chromatography (GC) method for their separation. Figure 2 (trace I) shows the efficiency of our GC analysis using a typical column for fatty acid separation of 60 m length and helium as the carrier gas. (for details, see Supporting Information). Unsatisfactory results were observed using hydrogen as carrier gas (data not shown). Indeed, we were aware of the possibility of peak superimposition with longer columns (100 m) and hydrogen as carrier gas;31 therefore, we checked for a good compromise between good resolution and reasonable elution times, foreseeing its application in a screening methodology. Two biological sources were used, namely, commercial human LDL samples and the cholesteryl ester fraction isolated from human plasma, the latter one obtained by a procedure free of oxidation and alteration of double bond geometry previously developed by us.36 The workup of biological samples for GC analysis requires the transesterification step to obtain fatty acid methyl esters (FAME). GC analysis of the fatty acid content of cholesteryl esters is shown in Figure 2 (trace II). The relative 1705



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percentages of the main 16:1 fatty acids recognized in the samples with the available references are listed in Table 1 (the complete fatty acid pattern is reported in Supporting Information, Tables S1−S3 and Figure S6). The natural cis isomers were present in the following relative percentages over the total content of fatty acids: 6-cis, 0.94 ± 0.23%; 9-cis, 1.74 ± 0.44%. Palmitelaidic acid was not detectable, whereas 6-trans16:1 was identified by comparison with the reference, although in a very low amount (0.05 ± 0.01%; see Figure 2, trace II). Quantitative determination gave the following results: 6-cis, 50.0 ± 4.0 ng/mL; 9-cis, 80.0 ± 7.0 ng/mL; and 6-trans, 3.0 ± 1.0 ng/mL. We considered that under our conditions other positional and geometrical fatty acid isomers could be present or superimposed to the identified peaks and that it was important to better investigate this aspect. Therefore, further examination by derivatization of the LDL and CE samples was carried out, as explained in the next section. For commercial human LDL samples, we followed two protocols: protocol 1 for the analysis of the total fatty acidcontaining lipids and protocol 2 for the separation of the lipid classes followed by the transesterification step using different methods for triglyceride (TG), cholesteryl ester (CE), and phospholipid (PL) fractions (see Experimental Procedures and Scheme S2, Supporting Information). TGs were found to be the minor components of LDL; however, the resolution of the GC chromatogram was still satisfactory. Representative GC runs of the separated TG, CE, and PL fractions of LDL are reported in Figure 2 (traces III, IV, and V), and Table 1 reports the 16:1 isomers recognized with the available references (the full analysis is reported in Supporting Information, Tables S1− S3; see also Figure S6). The quantitative analysis gave the following values in the total LDL lipids: 9-cis, 52 ± 2.0 ng/mL; 6-cis isomer, 35.0 ± 2.0 ng/mL; 9-trans isomer, 0.4 ± 0.1 ng/mL; and 6-trans isomer, 4.6 ± 0.3 ng/mL. These data, together with the above-reported ones, evidenced for the first time interesting variations of the 16:1 isomer content depending on the lipid classes and LDL fractions. Sapienic acid was found to be the major component of the TG fraction of LDL samples, whereas the prevalent positional isomer in the CE fraction of the LDL was palmitoleic acid, with a distribution similar to that of plasma CE. It is worth noting that trans isomers of other fatty acid families, such as C18:1, C18:2, and C20:4, were also individuated and quantified in the chromatograms (Tables S1−S3 and Figure S5, Supporting Information), confirming previously reported data.34,36,42 Analysis of Positional and Geometrical Isomers by Transformation into Their DMDS Adducts. The lipid extracts from LDL and plasma samples were treated to form dimethyl disulfide (DMDS) adducts, in order to ascertain unambiguously the presence of positional isomers, in particular the 6 and 9 isomers of hexadecenoic fatty acids. This is a wellknown procedure including the GC separation of the DMDS adducts and, more importantly, the examination of mass spectrometry fragmentation patterns, which are diagnostic of the double bond position.39,40,43 Scheme 1 shows the reaction of sapienic acid methyl ester, highlighting the two fragments expected for the DMDS adduct in the mass spectrometry (MS) analysis. However, DMDS adducts of the geometrical isomers (i.e., 6-cis and trans) have the same molecular masses and fragments (Scheme S1 and Figure S7, Supporting Information).

Scheme 1. Formation of the DMDS Adduct of Sapienic Acid Methyl Ester and Expected Fragmentation Pattern with the Molecular Weight of the Main Fragments

Examining the results of the LDL and plasma CE samples, we observed that the adduct corresponding to the double bond in the C6 position was clearly individuated with diagnostic fragmentations and molecular masses m/z of 187, 175, and 143 (175-CH3OH), as expected.39,40,43 Also, the adduct of the double bond in the 9 position was individuated by its characteristic fragmentations (Figure S7, Supporting Information). Moreover, careful investigation of the mass spectra of the DMDS adducts of the different lipid fractions was performed in order to ascertain the absence of mass fragments relative to positional isomers with the double bond in C7 and C843 and to exclude superimposition of peaks in the GC analysis. Indeed, we concluded that the 6 and 9 positional isomers are the main components individuated under our experimental conditions (Figure S8, Supporting Information).

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DISCUSSION The present results are relevant since plasma attracts enormous interest for its easiness of withdrawal and potential for lipidomic studies.44 The proposed chemical biology approach requiring analytical and synthetic chemistry is a good example of the multidisciplinary setting needed to address this important application for human plasma biomarker development (Scheme S2, Supporting Information). This approach is needed when minor positional and geometrical isomers are identified in a mixture with major fatty acids, before attributing them important roles of biomarker or signaling.11−17 Indeed, differences of double bond location and position in minor fatty acids can escape or cheat the detection, even when very sophisticated mass spectrometry tools are used. Cis 16:1 fatty acids in the 6 and 9 positions are known to be less connected to the diet, and their biosynthesis from saturated fatty acids (cfr, Figure 1) is expected to play a key role. In a study of the composition of human milk lipids, the 7 and 9 positional isomers were identified as the prevalent cis 16:1 isomers,31 whereas sapienic acid was not identified. However, in a recent report of the identification of sapienic acid in hair and nail samples, it is possible to see how critical the role of GC analytical conditions is to get a good resolution of the region corresponding to C16 fatty acids.25 Our protocol evidenced for the first time that with appropriate GC columns of 60 m length the separation is satisfactory for a full recognition of fatty acids in the samples (see Tables S1−S3, Supporting Information). It 1706



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cannot be evaluated in relationship with metabolic parameters. Further work is in progress to figure out the level and role of each isomer as well as its relationship with individual conditions. In conclusion, our results indicate the chemical biology approach as a powerful tool for the study of fatty acidcontaining lipids in biological samples when positional and geometrical isomers of minor fatty acids are concerned. Due to the important role of palmitoleic acid as a signaling molecule, this raises general attention to an accurate isomer attribution in biological samples. Indeed, the protocol and molecular library provided in this work were useful to discover sapienic acid as a relevant positional isomer in plasma together with its trans geometrical isomer. The practical significance and biological relevance of these findings will be addressed in further studies of the follow up of hexadecenoic fatty acid pathways and signaling under different health conditions, also in correlation with biomarker discovery in fatty acid-based lipidomics and the application for nutrilipidomic strategies.

is worth emphasizing that under our conditions helium gave better performance than hydrogen as the carrier gas (data not shown). Because the identification of fatty acids transferred to biological and clinical consequences, the analytical protocols play a key role in biomarker development. With the present work, the fundamental contribution of the correct identification of the C16 fatty acids is obtained, in view of the relevant activities attributed to palmitoleic acid so far, and motivates us to pay increased attention to other roles, still underestimated, played by sapienic acid. Interestingly, variations of sapienic and palmitoleic acid content in the lipid classes and LDL fractions were highlighted and deserve further studies in relationship with factors regulating FADS activities, as depicted in Figure 1. The hexadecenoic fatty acid presence in plasma and LDL cholesteryl esters (see Table 1) also suggests an interesting metabolic interplay with the activity of lecithin−cholesterol acyl transferase (LCAT), the enzymatic path transferring fatty acid tails from phospholipids to cholesterol.45 Sapienic acid was found as the major component of the TG fraction of LDL samples, whereas in the CE fraction of the LDL, the prevalent positional isomer was palmitoleic acid, clearly derived from PL by LCAT activity (Table 1 and Tables S1−S3, Supporting Information). This indicates different distribution of the hexadecenoic fatty acids among lipid classes, with sapienic acid prevalently in apolar lipids, which is in agreement with previous analysis of sebum triglyceride and wax ester fractions.22−24 From the fatty acid analysis (see Tables S1−S3, Supporting Information) another interesting difference emerges regarding the cis/trans ratios in the case of sapienic, palmitoleic, and oleic acids, with a higher ratio in the case of the former fatty acid. Again, this could be a reflection of the different locations of the fatty acids, suggesting that circulating apolar lipids containing sapienic acid are more exposed to free radical isomerizing conditions. The overall distribution and content of the MUFA family in correlation with specific metabolic conditions and the evaluation of these fatty acids as biomarkers are not yet investigated. The endogenous transformation of the cis hexadecenoic family to their corresponding trans geometrical isomers, involving fatty acids not prevalently derived from the diet, is an interesting path to be studied in different conditions in order to develop plasma biomarkers for fatty acid-based lipidomics, also involving radical stress.42 Trans isomers of palmitoleic and sapienic acids have been detected in our samples of LDL and plasma cholesteryl esters, quantitatively estimated at a total content of ca. 5 ng/mL, with a consistent contribution of transsapienic acid. So far, the isomers of 16:1 in human samples were attributed to the consumption of dairy products.31−33 In a very detailed analysis, human milk fat showed the content of several isomers (6-trans to 14-trans), whereas bovine milk fat had the trans isomers in the range 9-trans to 14-trans. Therefore, it was concluded that the 6-trans, 7-trans, and 8trans isomers could derive from the consumption of vegetable hydrogenated margarines or cooking fats.31 Our findings that sapienic acid exists in human plasma and that its trans isomer can be detected in comparison with the synthetically available reference suggest that the contribution of endogenous free radical isomerization of the natural and well represented cis fatty acid cannot be ruled out. The samples used in this work were not connected with dietary or health information on the human subjects; therefore, at this stage the fatty acid levels

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ASSOCIATED CONTENT

S Supporting Information *

Additional data and experimental details are given for the identification of the four hexadecenoic fatty acid isomers along with the calibration procedure. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

The support and sponsorship concerted by the COST Action CM1201 on “Biomimetic Radical Chemistry” are kindly acknowledged. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Lipinutragen Srl for providing blood samples from routine lipidomic analyses. ABBREVIATIONS Ag-TLC, silver-thin layer chromatography; CE, cholesteryl ester; DMDS, dimethyl disulfide; FADS, fatty acid desaturase; FAME, fatty acid methyl ester; GC, gas chromatography; LDL, low density lipoprotein; MUFA, monounsaturated fatty acid; NMR, nuclear magnetic resonance; PL, phospholipids; PUFA, polyunsaturated fatty acid; SCD1, stearoyl-CoA desaturase; TG, triglycerides

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REFERENCES

(1) Chatgilialoglu, C., and Ferreri, C. (2005) Trans lipids: the free radical path. Acc. Chem. Res. 38, 441−448. (2) Chatgilialoglu, C., Ferreri, C., Melchiorre, M., Sansone, A., and Torreggiani, A. (2013) Lipid geometrical isomerism: from chemistry to biology and diagnostics. Chem. Rev. DOI: dx.doi.org/10.1021/ cr4002287. (3) Mozaffarian, D., Katan, M. B., Ascherio, A., Stampfer, M. J., and Willett, W. C. (2006) Trans fatty acids and cardiovascular disease. N. Engl. J. Med. 354, 1601−1613. (4) Menaa, F., Menaa, A., Menaa, B., and Tréton, J. (2013) Transfatty acids, dangerous bonds for health? A background review paper of

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Hexadecenoic Fatty Acid Isomers: A Chemical Biology Approach for

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