Carbon-13-isotopomics and metabolomics of fatty acids from triacylglycerols: overcoming the limitations of GC-C-IRMS for short- and medium-acyl chains.

Carbon-13 isotopomics of triacylglycerol (TAG) fatty acids or free fatty acids in biological matrices holds considerable potential in food authentication, forensic investigations, metabolic studies, and medical research. However, challenges arise in the isotopic analysis of short- and medium-chain (C4 to C10) fatty acid methyl esters (SMCFAMEs) through gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). The high volatility of these esters results in losses during their preparation, leading to isotopic fractionation. Moreover, the methoxy group added to acyl chains requires the correction of δ13C values, thereby increasing the uncertainty of the final results. Analyzing free fatty acids (FFAs) addresses both issues encountered with SMCFAMEs. To achieve this objective, we have developed a new protocol enabling the isotopomics of individual fatty acids (FAs) by GC-C-IRMS. The same experiment also provides the FA profile, i.e., the relative percentage of each FA in the TAG hydrolysate or its concentration in the studied matrix. The method exhibited high precision, as evidenced by the repeatability and within-lab reproducibility of results when tested on TAGs from both animal and vegetal origins. Compared to the analysis of FAMEs by GC-C-IRMS, the current procedure also brings several improvements in alignment with the principles of green analytical chemistry and green sample preparation. Thus, we present a two-in-one method for 13C-isotopomic and metabolomic biomarker quantitation within quasi-universal TAG compounds, encompassing the short- and medium-acyl chains.

A precise and rapid isotopomic analysis of small quantities of cholesterol at natural abundance by optimized 1H-13C 2D NMR.

Cholesterol, the principal zoosterol, is a key metabolite linked to several health complications. Studies have shown its potential as a metabolic biomarker for predicting various diseases and determining food origin. However, the existing INEPT (insensitive nuclei enhanced by polarization transfer) 13C position-specific isotope analysis method of cholesterol by NMR was not suitable for very precise analysis of small quantities due to its long acquisition time and therefore is restricted to products rich in cholesterol. In this work, a symmetric and adiabatic heteronuclear single quantum coherence (HSQC) 2D NMR sequence was developed for the high-precision (few permil) analysis of small quantities of cholesterol. Adiabatic pulses were incremented for improving precision and sensitivity. Moreover, several strategies such as the use of non-uniform sampling, linear prediction, and variable recycling time were optimized to reduce the acquisition time. The number of increments and spectral range were also adjusted. The method was developed on a system with a cryogenically cooled probe and was not tested on a room-temperature system. Our new approach allowed analyzing as low as 5 mg of cholesterol in 31 min with a long-term repeatability lower than 2‰ on the 24 non-quaternary carbon atoms of the molecule comparing to 16.2 h for the same quantity using the existing INEPT method. This result makes conceivable the isotope analysis of matrices low in cholesterol. Graphical abstract.

A strategy for simultaneous determination of fatty acid composition, fatty acid position, and position-specific isotope contents in triacylglycerol matrices by 13C-NMR.

Triacylglycerols, which are quasi-universal components of food matrices, consist of complex mixtures of molecules. Their site-specific 13C content, their fatty acid profile, and their position on the glycerol moiety may significantly vary with the geographical, botanical, or animal origin of the sample. Such variables are valuable tracers for food authentication issues. The main objective of this work was to develop a new method based on a rapid and precise 13C-NMR spectroscopy (using a polarization transfer technique) coupled with multivariate linear regression analyses in order to quantify the whole set of individual fatty acids within triacylglycerols. In this respect, olive oil samples were analyzed by means of both adiabatic 13C-INEPT sequence and gas chromatography (GC). For each fatty acid within the studied matrix and for squalene as well, a multivariate prediction model was constructed using the deconvoluted peak areas of 13C-INEPT spectra as predictors, and the data obtained by GC as response variables. This 13C-NMR-based strategy, tested on olive oil, could serve as an alternative to the gas chromatographic quantification of individual fatty acids in other matrices, while providing additional compositional and isotopic information. Graphical abstract A strategy based on the multivariate linear regression of variables obtained by a rapid 13C-NMR technique was developed for the quantification of individual fatty acids within triacylglycerol matrices. The conceived strategy was tested on olive oil.

Biomarkers for cheese authentication by detailed and fast gas chromatographic profiling of triacylglycerol fatty acids.

Unsaturated fatty acid isomers and odd- and branched-chain fatty acids (OBCFAs) in milk triacylglycerols (TAGs) can be quantitated using gas chromatography (GC), providing access to biomarkers of animal species, breeds, diet, geographic origin, and environmental conditions. Such analysis requires expensive cyanopropyl siloxane or ionic liquid columns of at least 50 m in length, which increases the elution time. Aiming to use GC for cheese authentication and characterization while keeping the experiment time short and maintaining a good separation between fatty acid (FA) isomers, we considered using a 30 m polyethylene glycol-2-nitroterephthalate column. The FAs thus quantitated allowed the discovery of specific biomarkers for the origins of cheese varieties highly consumed in several countries. In addition, the simple and multivariate correlations we found between FAs in the cheese TAG matrix were alternative means for characterization and authentication purposes.

Cheese characterization and authentication through lipid biomarkers obtained by high-resolution 1H NMR profiling.

Food quality and safety are at the heart of consumers' concerns across the world. Dairy products, because of their large consumption, are fertile ground for fraudulent acts. This fact justifies the development of effective, accessible, and rapid analytical methods for their authentication. A high-resolution spectral treatment method previously developed by our team was applied to 1H NMR spectra of cheese triacylglycerols. 178 Peaks were thus quantitated and successfully used in the construction of multivariate models for the quantitation of individual fatty acids and for the classification of cheese samples according to the producing species, to their origin and variety. Besides, several peaks related to the amount and position of anteisopentadecanoic, butyric, α-linolenic, myristoleic, rumenic, and vaccenic acids were, among others, specific biomarkers of cheese groups. For the first time in 1H NMR, we were able to identify and to quantitate signals related to minor fatty acids within cheese triacylglycerols.

Acute limb ischemia and aortic mural thrombosis as primary manifestations of severe acute respiratory syndrome coronavirus 2.

A variable clinical presentation is emerging as a hallmark of the novel SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). In addition to hypoxic respiratory failure, multiorgan dysfunction, and septic shock, significant thromboembolic complications posited to result from diffuse coagulopathy have been associated with this viral infection. We report on a unique primary manifestation of SARS-CoV-2 infection presenting as acute limb ischemia and aortic mural thrombosis without clinical evidence of pulmonary disease. Despite our best attempts at limb salvage with therapeutic anticoagulation, emergent aortoiliac and distal embolectomy, the patient developed bilateral dry gangrene and ultimately required lower extremity amputations.

High-resolution 1H NMR profiling of triacylglycerols as a tool for authentication of food from animal origin: Application to hen egg matrix.

Metabolomics of complex biological matrices conducted by means of 1H NMR leads to spectra suffering from severe signal overlapping. Previously, we have developed a high-resolution spectral treatment method to help solving this issue in 1H NMR of triacylglycerols. In this work, we tested the potential of the developed method in the characterization and authentication of food products from animal origin using egg yolk as a model matrix. The approach consisted in a spectral deconvolution guided by the precision obtained on the deconvoluted peaks after reference lineshape adjustment of spectra. Thus, 135 peaks were quantitated and successfully used as biomarkers of origin, of hens breed, and of farming system. This required multivariate statistical analyses for classification. The same pool of variables allowed construction of multivariate quantitation models for individual fatty acids. Furthermore, minute amounts of conjugated fatty acids were quantitated and used as fingerprints of samples from backyard and free-range farming.

EBT3 Gafchromic® film as a new substrate for in vitro evaluation of sun protection factor.

There exist different methods for the determination of sun protection factor (SPF) values for sunscreens. We aimed to develop a new in vitro method using EBT3 Gafchromic® film as a substrate. The colour of EBT3 Gafchromic® film changes when exposed to UV light. Films were covered by sunscreen preparations of different SPF values ranging from 0 to 50. Uncovered and covered films were exposed to different solar light energies and their colour change was compared. Absorbance spectra of films was measured at 633 nm using a UV-VIS spectrophotometer apparatus. The colour of the film darkens when ultraviolet energy increases, which means that absorbance increases with exposure time. However, when films are covered by sunscreens, the colour change is less visible and the absorbance significantly decreases with increasing SPF value. There is a linear correlation between the absorbance of EBT3 Gafchromic® film and SPF value of sunscreens covering the film. Statistical analysis demonstrated that the SPF value of a sunscreen can be predicted using EBT3 Gafchromic® film as a substrate. This is the first report of an in vitro method based on colour change of a substrate which takes into consideration exposure time, and relates more closely to conditions of real-life. Based on these parameters, this is a reliable in vitro method for SPF testing.

Metabisotopomics of triacylglycerols from animal origin: A simultaneous metabolomic and isotopic profiling using 13C INEPT.

In previous works, we developed a 13C NMR method for analyzing triacylglycerols in olive oil using an adiabatic refocused INEPT sequence. This allowed spectral acquisition to be done in only 8 min with sufficient precision for isotopic measurements. In the present study, we made use of the same methodology to investigate the potential of triacylglycerols as source of biomarkers in animal origin matrices. To this end, egg yolk was taken as a model matrix. We called our profiling approach metabisotopomics since it was simultaneously metabolomic and isotopic profiling. Beside its ability to quantitate several fatty acids, metabisotopomics of triacylglycerols in egg yolk allowed the multivariate classification of samples according to the hen breed, to the farming system and origin. Achieved results confirmed our presumption that 13C metabisotopomics of triacylglycerols from animal sources is a powerful tool for metabolic studies as well as for food authentication processes.

Improved lipid mixtures profiling by 1H NMR using reference lineshape adjustment and deconvolution techniques.

ANALYSIS: of one-dimensional 1H NMR spectra of complex mixtures, such as lipids from natural extracts, is hampered by the small spectral width leading to a great number of overlapped signals. Additional complications including lineshape broadening and distortion may occur due to magnetic field inhomogeneity. Quantitation of such spectra is therefore challenging. We present in this work a quantitation approach based on deconvolution after correction of spectra by means of reference lineshape adjustment (RLA), also known as reference deconvolution. Spectral fit and precision obtained on deconvoluted peaks were used as indicators to iteratively improve the deconvolution process. This approach was tested on 1H NMR spectra of olive oil samples and allowed extraction of 77 peaks (available as peak intensities or areas), whereas spectral integration afforded 5 variables when only well-resolved signals were considered and 29 variables when a bucket around each discernible peak was integrated. Deconvoluted peak intensities and areas were obtained with improved precision after RLA of raw spectra. The use of these spectral variables as predictors in multivariate statistical analysis enhanced the classification of olive oil samples according to the altitude of the olive field or to the color of the olive drupes. The same variables allowed quantitation of oleic, palmitoleic, and vaccenic acids within triacylglycerols, which was not possible by 1H NMR, and improved quantitation of linoleic and linolenic acids. These results proved the high potential of the presented approach in the characterization and authentication of complex mixtures by 1H NMR spectroscopy.

Cholesterol, a powerful 13C isotopic biomarker.

Cholesterol is related to many health diseases and is considered as a metabolic disorder biomarker. This compound, present in all food products of animal origin, can also be used as food authentication biomarker. In this work and for the first time, positional 13C isotope contents were determined for such a high molecular weight compound. This was possible by means of NMR using adiabatic refocused INEPT. In order to test the potential of this approach for discrimination, hen eggs from different origins were collected. Quantitative extraction of egg yolk cholesterol was optimized, and partial reduced molar fractions of its different 13C isotopomers were used as predictors in discriminant analysis. Compared with the global 13C isotopic composition determined using isotope ratio monitoring by Mass Spectrometry, the relative content of cholesterol 13C isotopomers added valuable power to sample classifications according to their origins. This study paves the way to isotopomics of other steroids and similar molecular weight compounds.

Origanum essential oils reduce the level of melanin in B16-F1 melanocytes.

BACKGROUND: Hyperpigmentation disorders are considered signs of skin aging and are aesthetically unpleasant. Most active ingredients used against hyperpigmentation disorders predominantly target tyrosinase activity. OBJECTIVES: To study the effect of two Origanum essential oils on the melanogenic activity of B16-F1 murine melanocytes. The main component of these oils, carvacrol, was also investigated and a model for anti-melanogenic activity is proposed. MATERIALS AND METHODS: B16-F1 melanocytes were exposed to different concentrations of essential oils and carvacrol. The level of tyrosinase and melanin was determined using spectrophotometric measurements. RESULTS: Essential oils of Origanum syriacum and Origanum ehrenbergii led to a significant 14% and 17% reduction in melanin level at 40 µg mL-1, respectively. However, neither demonstrated a significant effect on the level of intracellular tyrosinase. The same effects were found for carvacrol which led to a 30% reduction in melanin at 45 µg mL-1. CONCLUSION: Our results indicate that the oils studied are anti-melanogenic. We propose a mechanism, similar to that for hydroquinone, whereby carvacrol functions as a competitive inhibitor of tyrosinase, thus inhibiting oxidation of tyrosine and causing a deregulation of melanogenesis.

Phytochemical screening and antityrosinase activity of carvacrol, thymoquinone, and four essential oils of Lebanese plants.

OBJECTIVE: In our study, we aim to explore the ability of four essential oils (EO) of Lebanese plants to inhibit the tyrosinase activity and to correlate their efficiency level to their phytochemical compositions. METHODS: The EO have been extracted by hydrodistillation using a Clevenger apparatus and have been studied by GC-MS analysis. Active compounds of Origanum species were identified and antityrosinase activities of EO and active molecules (carvacrol and thymoquinone) have been tested in tubo. RESULTS: Antityrosinase activities were obtained as follows: EO of Origanum syriacum (80.41% ± 2.00%), EO of Origanum ehrenbergii (45.33% ± 2.20%), EO of Salvia fruticosa (14.62% ± 2.30%), EO of Calamintha origanifolia (16.51% ± 5.80%), Carvacrol (56.55% ± 3.10%), and Thymoquinone (19.49% ± 1.50%). CONCLUSION: Origanum essential oils resulted in the highest antityrosinase activity due to their high content in carvacrol. However, when present together with carvacrol, thymoquinone decreases the efficiency of carvacrol, which is the case of O. ehrenbergii essential oil. Thus, for improved antityrosinase activity, O. syriacum and O. ehrenbergii should be harvested during flowering stage where carvacrol is present at its highest dosage and thymoquinone at its lowest.

Fungal contamination and Aflatoxin B1 and Ochratoxin A in Lebanese wine-grapes and musts.

Five hundred and ten strains of filamentous fungi were isolated from Lebanese grapes during 2005 at veraison and harvesting periods. Four hundred eighty-seven isolates belonged to the Aspergillus spp. (95.5%) and 23 belonged to the Penicillium spp. (4.5%). Black aspergilli constituted 56.9% (52.2% Aspergillus niger aggregates, 2.9% Aspergillus japonicus and 1.8% Aspergillus carbonarius) while the isolation rate of Aspergillus flavus the none habitual member of grape mycobiota was 43.1% of the total Aspergillus spp. isolated. All isolates were tested for the ability to produce the Ochratoxin A (OTA) and the Aflatoxin B1 (AFB1). A. carbonarius showed that it is the only species able to produce the OTA with a production ability of 100% and a maximum concentration reaching 8.38microg/g CYA. As for the aflatoxigenic ability, 43.4% of A. flavus isolates produced this mycotoxin with a maximum production reaching 22.6microg/g CYA while none of the other isolates showed a production capacity of this mycotoxin. Forty-seven samples of must produced from the collected grapes were also analyzed. None of these samples was contaminated by OTA at a detectable limit while 40% of these same samples were found to contain AFB1 with concentrations ranging from 0.01 to 0.46microgl(-1).

Olive oil characterization and classification by 13C NMR with a polarization transfer technique: A comparison with gas chromatography and 1H NMR.

In a previous work, we optimized and used a fast adiabatic 13C-INEPT (Insensitive Nuclei Enhanced by Polarization Transfer) experiment for the isotopomic analysis of olive oil samples, which allowed us quantifying individual fatty acids within triacylglycerols through multivariate linear regression models. The goal of this study was to validate these models and to evaluate the power of 13C-INEPT in the authentication of olive oils relative to gas chromatography (GC) and 1H NMR. In this respect, a new set of olive oil samples was analyzed by these three techniques. The analytical variables thus obtained as well as their corresponding long-term repeatability were compared. As a result, the reliability of the fatty acid quantification models was proven and the best classification of olive oils according to the altitude of the olive grove and to the morphological aspect (color) of the olives was achieved by means of 13C-INEPT.

Structure, gelation, and antioxidant properties of curcumin-doped casein micelle powder produced by spray-drying.

The encapsulation of curcumin in micellar caseins (MCs) and the production of powder were performed by spray-drying. Nearly 97% of the curcumin was retained and the yellow powder showed a typical high casein powder morphology. The hygroscopic properties were determined, slight differences reflected less available hydrophobic sites when curcumin was bound to casein, favoring interactions with water in curcumin-enriched MC powders. No difference was detected on the internal MC structure via SAXS. The antioxidant activity of doped-curcumin powder presented 88% of active curcumin. For 60 days at 40 °C storage, the antioxidant activity of curcumin measured by ABTS and FRAP assays was preserved with a percentage of 82 ± 2.0% and 84 ± 1.1%, respectively. Curcumin doped powders presented similar features to classical casein powders (rehydration and gelling abilities). It was demonstrated that curcumin encapsulation in MCs in its powder form helped in protecting its antioxidant activity without influencing the techno-functional properties of MCs. This study allowed the incorporation of curcumin via the MC matrix as an active food ingredient available in a powder state usable as classical milk powder in several food formulations.

Geoclimatic, morphological, and temporal effects on Lebanese olive oils composition and classification: A (1)H NMR metabolomic study.

Two hundred and thirty-four Lebanese olive samples were collected from different regions and the corresponding oils were analysed by (1)H NMR spectroscopy. The variables obtained, related to fatty acids and minor components, were used as inputs in univariate and multivariate analyses aiming to characterize and classify the oils according to geographical, morphological, and temporal factors. Samples were sorted according to the colour, size, and shape of olives, which allowed statistically significant classifications to be achieved. A sequential strategy was developed to discriminate among samples from different altitudes and latitudes. Following this strategy, obvious trends and classifications were obtained at subregional level. Furthermore, the shift in the harvest date within a range of three weeks was considered and its effect on the classification models was investigated. Likewise, the harvest year effect was evaluated; the precipitation level in April and May had a significant impact on the characteristics of the oils.

Occurrence of ochratoxin A- and aflatoxin B1-producing fungi in Lebanese grapes and ochratoxin a content in musts and finished wines during 2004.

This paper reports the results of an extensive survey on the occurrence of filamentous fungi isolated from wine-grapes in Lebanon and to test their ability to produce ochratoxin A (OTA) and aflatoxin B1 (AFB1) on CYA culture medium, in order to assess their potential for producing these mycotoxins on grapes. From the 470 grapes samples taken during season 2004, 550 fungi strains were isolated with 490 belonging to Aspergillus spp. and 60 belonging to Penicillium spp. All these isolated fungi starins were tested for their ability to produce OTA and AFB1. Aspergillus carbonarius shows that it is the only species able to produce OTA with a production percentage reaching 100% and a maximum concentration of 52.8 microg/g of Czapek yeast extract agar (CYA). In its turn, Aspergillus flavus was considered as the only AFB1-producing species with production percentage of 45.3% and a maximum concentration reaching 40 microg/g CYA. A total of 47 handmade musts produced from the collected grapes were also analyzed in order to correlate the presence of OTA in must and the occurrence of filamentous fungi on grapes; 57.4% were contaminated with OTA at low level with concentrations ranging between 0.011 and 0.221 microg OTA L(-1). The analysis of these must samples was not performed with regard to AFB1. Seventy samples of finish red wine were also assayed for OTA content. The results showed that 42 of the tested samples (60%) were found to be positive for OTA with low levels (0.012-0.126 microg OTA L(-1)).

Precise and rapid isotopomic analysis by (1)H-(13)C 2D NMR: Application to triacylglycerol matrices.

An optimized HSQC sequence was tested and applied to triacylglycerol matrices to determine their isotopic and metabolomic profiles. Spectral aliasing and non-uniform sampling approaches were used to decrease the experimental time and to improve the resolution, respectively. An excellent long-term repeatability of signal integrals was achieved enabling to perform isotopic measurements. Thirty-two commercial vegetable oils were analyzed by this methodology. The results show that this method can be used to classify oil samples according to their geographical and botanical origins.

Structure and gelation properties of casein micelles doped with curcumin under acidic conditions.

In this study, the ability of micellar casein (MC) to interact with curcumin during acidification and to produce acid gel was investigated. Steady-state fluorescence spectroscopy of curcumin variation and fluorescence quenching of caseins upon binding with curcumin molecules were evidenced. Increasing the temperature from 20 to 35 °C enhanced MC-curcumin interactions as reflected by the increase in the binding constant from 0.6 ± 0.3 × 10(4) to 6.6 ± 0.6 × 10(4) M(-1). From changes in entropy, enthalpy and Gibbs free energy, hydrophobic interactions were proposed as major binding forces. Static fluorescence MC quenching was demonstrated for the MC-curcumin complex during acidification. From pH 7.4 to pH 5.0, the binding site numbers varied in the range from 1.25 ± 0.05 to 1.49 ± 0.05 and the binding constant kb varied from 3.9 ± 0.4 × 10(4) to 7.5 ± 0.7 × 10(4) M(-1). Small angle X-ray scattering profiles demonstrated that the MC internal structure was unchanged upon curcumin binding. The ζ-potential value of curcumin-doped MC indicated that curcumin did not modify the global charge of MC particles. Acid gelation studied by oscillation rheology and static multiple light scattering at 20 and 35 °C led to a similar behavior for native and curcumin-doped MC suspensions. For the first time, it was demonstrated that the colloidal and functional properties of MC were unchanged when doped with curcumin during acidification.