Data of detection and characterization of nitrated conjugated-linoleic acid (NO2-cLA) in LDL

Under physiological and pathophysiological conditions, lipid nitration occurs generating nitro-fatty acids (NFA) with pleiotropic activities as modulation of inflammatory cell responses. Foam cell formation and atherosclerotic lesion development have been extensively related to low-density lipoprotein (LDL) oxidation. Considering our manuscript “Fatty acid nitration in human low-density lipoprotein” (https://doi.org/10.1016/j.abb.2019.108190), herein we report the oxidation versus nitration of human LDL protein and lipid fractions. Data is shown on LDL fatty acid nitration, in particular, formation and quantitation of nitro-conjugated linoleic acid (NO2-cLA) under mild nitration conditions. In parallel to NO2-cLA formation, depletion of endogenous antioxidants, protein tyrosine nitration, and carbonyl formation is observed. Overall, our data propose the formation of a potential anti-atherogenic form of LDL carrying NFA.


Data description
Special focus on nitration of cLA was done since previous reports found that this fatty acid is the most susceptible to nitration under physiological conditions. First, we measured cLA content in LDL samples by derivatization with PTAD on the free fatty acid fraction of LDL (Fig. 1A). Since PTAD reacts with conjugated double bonds, the 9,11-cLA standard was employed for the construction of an external calibration curve in order to quantitate cLA in LDL (Fig. 1B). The mean content of total cLA (free and esterified) on LDL samples (Fig. 1C) was 44 pmol/mg apoB100.
Considering that cLA is present on the LDL lipidic fraction and is susceptible to nitration, we next analyzed LDL samples treated with constant low fluxes of peroxynitrite. Fig. 2 shows a representative chromatogram of HPLC-MS/MS analysis in which peaks corresponding to NO 2 -cLA were found in peroxynitrite-oxidized LDL. The HPLC method separates and identifies with high accuracy different isomers of NFA, e.g. NO 2 -LA and NO 2 -cLA. The identity of NO 2 -cLA was confirmed by comparing the retention times with the NO 2 -LA and NO 2 -cLA standards ( Fig. 2A). No significant amounts of other NFA were detected (data not shown). Additional experiments were performed to confirm the presence of a nitroalkene group on the detected compounds. For this purpose, samples were incubated with BME to allow Michael addition reaction between the free thiol and the electrophilic moiety in the NFA. Afterward, samples were re-analyzed and peaks assigned to NO 2 -cLA-were completely reduced (Fig. 2B).

Value of the Data
The data show the quantification of conjugated-linoleic acid (cLA) in LDL and its nitration by peroxynitrite fluxes.
Our data presented in the article show how constant fluxes of peroxynitrite nitrate cLA in LDL in parallel to a-tocopherol depletion, apoB-100 tyrosine nitration, and protein carbonyl formation. Studies of LC-MS/MS, HPLC-Fluorescence detection, and dot blot were done to obtain the data presented in the article, being useful tools for the detection of circulating NFA-loaded LDL in plasma samples. Researchers studying lipid modifications and metabolism related to oxidative and inflammatory processes or chemical and analytical characterization of compounds with biological relevance may take advantage of the data set presented in this work. LDL was exposed to oxidation and the nitrating system as explained before. Dot-blot analysis of carbonyls was assessed as a fingerprint of protein oxidation. Peroxynitrite induced a hyperbolic increase in carbonyl formation in a dose-dependent manner with a saturation of carbonyl formation at 1.2 mM (Fig. 3A). As expected, a-TOH was rapidly oxidized from LDL being depleted after 10 min (Fig. 3B). All samples were submitted to the dot-blot analysis of 3-nitrotyrosine (3-NT) formation. Peroxynitrite showed a hyperbolic-like increase in 3-nitrotyrosine formation in a dose-dependent manner. Peroxynitrite infusion reaches a maximum of 3-NT formation at 2 mM (Fig. 3C).
Finally, the kinetics of LDL oxidation were compared in parallel with NFA formation. NO 2 -cLA quantitation overtime during peroxynitrite infusion was performed (Fig. 3D). The nitration curve of cLA is steepest at initial stages of incubation until it reaches a maximum at 45 min when a-TOH was totally depleted and protein oxidation almost reached a plateau. Under these experimental conditions, a mean value of 5 pmol of NO 2 -cLA/mg of apoB-100 can be found, which represents about 10% of total cLA.

LDL purification
Low-density lipoprotein (LDL) was purified from healthy normolipidemic donors as previously reported [1]. The absence of other plasmatic proteins in the LDL fraction was verified by agarose gel electrophoresis and apoB-100 protein concentration was obtained by absorbance at 280 nm (ε ¼ 1.05 (mg/mL) À1 . cm À1 ) [2].

LDL oxidation
LDL nitration was performed in 100 mM potassium phosphate buffer, pH 7.3 with 100 mM DTPA. Peroxynitrite was added for 60 min to 3 mM LDL as a continuous infusion of 20 mM/min. Due to the strong alkaline solution of peroxynitrite, the pH of all samples was checked at the end of the treatment and stayed within a 0.2 pH unit range. Reverse addition control was performed in all cases by the previous decomposition of peroxynitrite in phosphate buffer pH 7.3. After incubation with peroxynitrite, 0.025% BHT was added in order to prevent further oxidation reactions and samples were then analyzed [1,3]. Transitions followed in each condition are indicated in the panels. B) The electrophilic reaction with bME was evaluated to confirm electrophilic NO 2 -cLA transitions.

Lipid analysis
. LDL samples (3 mM, 500 mL) were oxidized with peroxynitrite as explained above. Then, lipoprotein samples were incubated with pancreatic lipase (5 mg) and phospholipase A1 (40 U) in potassium phosphate buffer 50 mM, pH 7.4 at 37 C under constant stirring for 1 hour. Triglyceride and phospholipid hydrolysis was followed by thin-layer chromatography [4]. After enzymatic treatment, lipids were extracted with hexane and the organic phase was separated, evaporated to dryness and resuspended in chloroform. Prior to lipid extraction, a mixture of nitrated fatty acids (NFA) internal standards -[ 13 C] 18 NO 2 -LA and 15 NO 2 -cLA-was added to each sample. A solid-phase extraction method with StrataNH2 cartridges (55 mm, 500 mg/6 mL, Phenomenex) was performed to obtain a fraction, eluted from the column with methanol, enriched in non-esterified fatty acids [5e8].

Conjugated linoleic acid (cLA) detection
Dried unesterified fatty acid fractions were resuspended in an acetonitrile solution containing 10 mM of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). This compound rapidly reacts with conjugated dienes and the resulting cLA-PTAD derivatives are detected and quantified by LC-ESI-MS/MS in the positive ion mode (m/z 438/178) [5]. B) MeOH extraction and reversed-phase HPLC quantitation of a-tocopherol, using a fluorescence detector (lex ¼ 295 nm, lem ¼ 330 nm). A representative chromatogram is shown on inset; C) dot-blot analysis employing polyclonal anti-3-NT antibodies. Representative primary data is shown on inset; D) Quantitation of NO 2 -cLA was performed by LC-MS/MS employing internal standards and a calibration curve. Data are shown from three independent experiments with n ¼ 3. A, B, C, data is relative to maximum signal and represents mean ± SD. D, Data are related to apoB-100 content in samples and represent mean ± SD.

Nitro-fatty acid detection
The methodology employed for NFA detection and quantitation was performed by HPLC-MS/MS using a triple quadrupole with a linear ion trap mass spectrometer (QTRAP4500, ABSciex) in the MRM mode [5,9,10].

b-Mercaptoethanol (BME) treatment
Dried unesterified fatty acid fractions were resuspended in 100 mM potassium phosphate buffer, pH 7.3 containing 2 mM BME, and incubated at 37 C for 1 hr. This thiol-compound rapidly reacts with nitroalkenes forming covalent adducts with retention times and m/z different than their precursors [5,6,8,11].

a-Tocopherol (a-TOH) depletion
a-Tocopherol content was determined by RP-HPLC using an 1100 Agilent quaternary pump HPLC with a fluorescent detector. LDL samples were mixed with cold methanol (1:9 v:v) and centrifuged at 14000 g for 20 min; the supernatant was injected into a reverse phase C18 column (Supelco, 250 mm Â 4.6 mm, particle size 5 mm) and a-TOH was eluted with MeOH and detected by fluorescence (lex ¼ 295 nm; lem ¼ 330 nm) [12].

Proteins' carbonyls
Carbonyls protein detection and quantitation were performed by using the DNPH derivatization method. Samples were transferred to PVDF membranes and DNPH derivatization was performed in HCl 2 N. After washing and blocking the membrane, anti-DNP antibodies were used to detect carbonyls [13]. Quantitation was performed by densitometry analysis employing Oddysey Li-Cor software.