Quantification Of Fatty Acids Research Articles

Identification and quantification of branched-chain fatty acids and odd-chain fatty acids of mammalian milk, dairy products, and vegetable oils using GC/TOF-MS

Branched-chain fatty acids (BCFAs) and odd-chain fatty acids (OCFAs) in mammalian milk, dairy products, and vegetable oils were investigated; 72 fatty acids, including 16 BCFAs and 10 OCFAs, were identified and quantified using GC/TOF-MS. Human milk showed the lowest content of BCFA (0.4% of total FA) and the content in other milk ranged from 2 to 6%, of which ai17:0, i17:0, ai15:0 and i15:0 were most abundant. Differences in the content of OCFA in mammalian milk trended similarly to differences in BCFA content. The molecular species of BCFA in cow milk-derived dairy products were identical, but the content was different, with higher content in fermented dairy products. Apparent differences in BCFA and OCFA composition were found in infant formulas, strongly related to their fat sources. Vegetable oils contained no BCFA and a small amount of OCFA. A scientific basis for further investigation of high-BCFA and OCFA dairy products is provided.

Sensitive quantification of short-chain fatty acids combined with global metabolomics in microbiome cultures.

The microbiome has been identified to have a key role for the physiology of their human host. One of the major impacts is the clearance of bacterial pathogens. We have now developed a chemoselective probe methodology for the absolute quantification of short-chain fatty acids at low nM concentrations, with high reproducibility and spiked isotope labelled internal standards. Immobilization to magnetic beads allows for separation from the matrix and the tagged metabolites upon bioorthogonal cleavage can be analyzed via UHPLC-MS. The major advantage of our sensitive method is the simple combination with global metabolomics analysis as only a small sample volume is required. We have applied this chemical metabolomics strategy for targeted SCFA analysis combined with global metabolomics on gut microbiome co-cultures with Salmonella and investigated the effect of antibiotic treatment.

Preliminary Analysis of Unsaturated Fatty Acid Profiles of Coffea arabica L., in Samples with a Denomination of Origin and Speciality of Oaxaca, Mexico.

In February 2020, Coffea arabica L. grown on the coast and in the Southern Sierra Madre of the state of Oaxaca, Mexico obtained the denomination of origin. Which does not have data on color and chemical composition, the first associated with the degree of roasting and the second with lipids (17-18%), as the group of compounds responsible, in part, for flavor, consistency, and may contribute to health benefits. In the present work, color was determined on the CIE L*a*b* scale and the unsaturated fatty acids by Nuclear Magnetic Resonance (NMR) of 1H and 13C in samples of medium roasted specialty coffee from the "Pluma" coffee-growing region, Oaxaca, Mexico. The average value of L* luminosity in ground coffee was 42.1 ± 0.1 reported for a light roast. Unsaturated fatty acids were quantified from the lipid fraction of the gr1 ound grain by NMR 1H and 13C, obtaining on average the highest abundance of linoleic (41.7 ± 0.5 by 1 H and 41.24 ± 0.5 by 13C), followed by oleic (9.2 ± 0.2 by 1H and 7.4 ± 0.2 by 13C) and linolenic (1.5 ± 0.1 by H and 1.1 ± 0.2 by 13C). This study indicates that 1H and 13C NMR spectroscopy is a useful tool for the quantification of linolenic, linoleic, and oleic fatty acids by the method of key signal shifts of these acids found in lipid samples in roasted coffee grains.

Green synthesis of gardenia seeds-based carbon dots for bacterial imaging and antioxidant activity in aqueous and oil samples.

In this work, luminescent carbon dots with gardenia seeds as carbon precursors (GCDs) were synthesized using a one-step mild pyrolysis process and were then used as probes for imaging of bacterial (Escherichia coli). The GCDs showed a strong emission at 430 nm when excited at 370 nm. The relative fluorescence quantum yield of GCDs was found to be 1.13% in an aqueous medium. Rapid internalization of the GCDs by bacteria was confirmed by three colors (blue, green, and yellow) images that were obtained using confocal fluorescence microscopy. In addition, GCDs were noted to exhibit potent scavenging activities against DPPH˙, ˙OH, and ˙O2- free radicals. GCDs were also assayed as antioxidants in an oil sample by volumetric determination of the peroxide value. Thus, GCDs exhibited good antioxidant properties both in aqueous and oil media. In addition, a free fatty acid quantification kit in the presence of GCDs showed enhanced fluorescence detection of palmitic acid with a remarkably good limit of detection of 0.08 μM, which is lower than that in the absence of GCDs (0.76 μM). The proposed fluorescence method was then successfully used to determine the concentration of palmitic acid spiked in milk powder samples, with spiked recoveries of 82.6-109.6% and relative standard deviations of 0.9-4.6%.

Processing induced trans fatty acid quantification analysis of selected edible oils and their probable outcome

Processing induced trans fatty acid quantification analysis of selected edible oils and their probable outcome

In Vitro Assessment of the Prebiotic Potential of Xylooligosaccharides from Barley Straw.

Barley straw was subjected to hydrothermal pretreatment (steam explosion) processing to evaluate its potential as a raw material to produce xylooligosaccharides (XOS) suitable for use as a prebiotic. The steam explosion pretreatment generated a liquid fraction containing solubilised hemicellulose. This fraction was purified using gel permeation chromatography to obtain a fraction rich in XOS DP2-DP6. The sample was characterised through analytical techniques such as HPAEC-PAD, FTIR and MALDI-TOF-MS. The prebiotic activity was evaluated using in vitro fermentation in human faecal cultures through the quantification of short-chain fatty acid (SCFA) and lactate production, the evolution of the pH and the consumption of carbon sources. The total SCFA production at the end of fermentation (30 h) was 90.1 mM. Positive significant differences between the amount of XOS from barley straw and fructooligosaccharides after incubation were observed.

Chemical Profile and Antimicrobial Activity of Syagrus oleracea (Mart.) Becc. Oils and Oils Extracted from Speciomerus revoili (Pic.) Larvae

Pharmacognosy Research,2023,15,1,71-74.DOI:10.5530/097484900265Published:December 2022Type:Original ArticleAuthors:Luciana Helena Veloso, Dario Alves de Oliveira, Cintya Neves de Souza, Francine Souza Alves, Vany Ferraz, Anna Christina de Almeida, Afrânio Farias de Melo Júnior, Elytania Veiga Menezes, Murilo Malveira Brandão, and Vanessade Andrade Royo Author(s) affiliations:Luciana Helena Veloso1, Dario Alves de Oliveira1, Cintya Neves de Souza2, Francine Souza Alves2, Vany Ferraz3, Anna Christina de Almeida2, Afrânio Farias de Melo Júnior1, Elytania Veiga Menezes1, Murilo Malveira Brandão1, Vanessade Andrade Royo1,* 1Program of Postgraduate Biotechnology, State University of Montes Claros (UNIMONTES), Montes Claros, MG, BRAZIL. 2Institute of Agricultural Sciences, Federal University of Minas Gerais (UFMG), Montes Claros, MG, BRAZIL. 3Department of Chemistry, Chromatography Laboratory, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG, BRAZIL. Abstract:Background: The antimicrobial effect of oils extracted from Syagrus oleracea (Mart.) to prove the action and obtain data for the future development of therapeutic products. Objectives: The present study was to determine the chemical profile and the antimicrobial potential of oils extracted from Syagrus oleracea (Mart.) Becc. almonds and Speciomerus revoilie (Pic.) larvae to obtain scientific information to prove the action and obtain data for the future development of therapeutic products. Materials and Methods: The separation, identification and quantification of fatty acids in oils extracted from the larvae of S. revoili (Coleoptera, Chrysomelidae) and almonds of S. oleracea (Arecaceae) were performed by gas chromatography coupled with mass spectrometry. The determination of antibacterial activity was performed using microdilution in plates. Results: In the oils extracted from S. revoili and S. oleracea, saturated chain fatty acids, the presence of hexanoic acid (only in almonds), and caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid and those of unsaturated chain, oleic acid (omega-9) and linoleic acid (omega-6). S. revoili oil showed satisfactory results against Escherichia coli and Staphylococcus aureus. Keywords:Antimicrobial activity., Fatty acids, Speciomerus revoili, Syagrus oleraceaView:PDF (138.34 KB)

Quantification of free and esterified long-chain fatty acids without extraction by high-performance liquid chromatography in anaerobic digestion sludge

Quantification of free and esterified long-chain fatty acids without extraction by high-performance liquid chromatography in anaerobic digestion sludge

Free Fatty Acids from Type 2 Diabetes Mellitus Serum Remodel Mesenchymal Stem Cell Lipids, Hindering Differentiation into Primordial Germ Cells.

Type 2 diabetes mellitus (T2DM) adversely affects the essential characteristics of adipose tissue-derived mesenchymal stem cells (AdMSCs). Given that T2DM is associated with an altered serum free fatty acid (FFA) profile, we examined whether diabetic serum FFAs influence the viability, differentiation, and fatty acid composition of the major lipid fractions of human AdMSCs in vitro. Serum FFAs were isolated from 7 diabetic and 10 healthy nondiabetic female individuals. AdMSCs were cultured and differentiated into primordial germ cell-like cells (PGCLCs) in the presence of either diabetic or nondiabetic FFAs. Cell viability was assessed using trypan blue staining. Cell differentiation was evaluated by measuring the PGCLC transcriptional markers Blimp1 and Stella. Lipid fractionation and fatty acid quantification were performed using thin-layer chromatography and gas-liquid chromatography, respectively. Both diabetic and nondiabetic FFAs significantly reduced the viability of PGCLCs. The gene expression of both differentiation markers was significantly lower in cells exposed to diabetic FFAs than in those treated with nondiabetic FFAs. Saturated fatty acids were significantly increased and linoleic acid was significantly decreased in the cellular phospholipid fraction after exposure to diabetic FFAs. In contrast, monounsaturated fatty acids were reduced and linoleic acid was elevated in the cellular triglyceride fraction in response to diabetic FFAs. Such an altered serum FFA profile in patients with T2DM reduces the proliferation and differentiation potential of AdMSCs, presumably due to the aberrant distribution of fatty acids into cell phospholipids and triglycerides.

English

The study analysed the fatty and amino acid compositions of chia seeds and the physicochemical characterisation of chia seeds oil. The quantification of amino and fatty acids was done using high-performance liquid chromatography (HPLC) and gas chromatography equipped with a flame ionisation detector (GC-FID). The amino acids from chia seeds were leucine, valine, phenylalanine, lysine, isoleucine, threonine, histidine, and methionine with 1.63, 1.37, 1.34, 1.14, 0.98, 0.98, 0.88 and 0.81 g/100 g, respectively. Saturated and unsaturated fatty acids were 3.95 and 30.48%, respectively. Meanwhile, the mean ratio of Omega 6/Omega 3 fatty acids was 0.333. The established physicochemical characteristics of CSO were acid value 2.63 mg KOH/g, peroxide value 6.23 meq active oxygen/kg, iodine value 200.721 g I2/100 g, saponification value 193.345 mg KOH/g, refractive index 1.454 at 40°C relative density 0.8824 g/cm3 at 20°C and specific gravity 0.882 at 40°C. The fatty acid and amino acid profiling of chia seeds revealed the presence of an appreciable amount of polyunsaturated fatty acids (PUFA) and essential amino acids. The physicochemical characteristics of CSO constitute a drying oil category that requires timely usage and proper storage condition to reduce rancidity from air or light exposure.   Key words: Chia seeds, chia seeds oil, amino acids, fatty acids, physicochemical characteristics.

Identification and quantification of fatty acids and lipid-soluble phytochemicals using GC-MS, HPLC-MS, and FTIR and their association with quality parameters during avocado ripening.

Consumer demand for the avocado fruit has increased considerably, but accelerated fruit ripening, lack of fruit ripening uniformity, and lack of proper quality characteristics and indices generate considerable problems during fruit handling and trade. Physicochemical parameters are used to determine avocado fruit ripening. These parameters together with lipid-soluble phytochemicals (LSP) and fatty acids (FAs) highlight the health and economic importance of this fruit. Analysis of LSP and FAs in avocado fruit has been reported, but combining the use of analytical techniques such as Fourier transform infrared spectroscopy (FTIR), gas chromatography-mass spectrometry (GC-MS), and high-pressure liquid chromatography coupled to mass spectrometry (HPLC-MS) to determine qualitative and quantitative changes during fruit ripening, and their association with physicochemical parameters, has not been conducted. Physicochemical parameters (fruit firmness, color, oil and dry matter contents) were determined, and the changes of FAs and LSP (carotenoids, chlorophylls, and tocopherols) during "Hass" avocado ripening, based on dry matter (DM) content, were analyzed using FTIR, GC-MS, and HPLC-MS. The association between them was also determined using principal component analysis. Fruit with 19% and 25% DM exhibited high LSP and FAs levels. Carotenoids, chlorophylls, and tocopherols were associated with firmness, color, and DM of 19% and 22%, while FAs were associated with fruit weight and DM of 25%. PRACTICAL APPLICATION: There is a major global increase in avocado cultivation and consumer demand. However, a major problem facing the handling and trade of avocado fruit is related to lack of fruit ripening uniformity and quality characteristics and indices. Therefore, a proper association between simple measures of fruit physicochemical properties and bioactive components can establish an excellent, simple, and practical index that can eventually be used for quality evaluation by the industry and the consumers.

Alpinumisoflavone Exhibits the Therapeutic Effect on Prostate Cancer Cells by Repressing AR and Co-Targeting FASN- and HMGCR-Mediated Lipid and Cholesterol Biosynthesis.

Prostate cancer (PCa) is the most common cancer in men, and this has been mainly noticed in Western and Asian countries. The aggregations of PCa and castration-resistant PCa (CRPC) progression are the crucial causes in the mortality of patients without the effective treatment. To seek new remedies for the lethal PCa diseases is currently an urgent need. In this study, we endeavored to investigate the therapeutic efficacy of alpinumisoflavone (AIF), a natural product, in PCa. LNCaP (androgen- sensitive) and C4-2 (CRPC) PCa cells were used. An MTT-based method, soft agar colony forming assay, biological progression approaches were applied to determine cell viability, migration, and invasion. A fatty acid quantification kit, a cholesterol detection kit and oil red O staining were conducted to analyze the intracellular levels of lipids and cholesterols. Apoptosis assays were also performed. AIF reduced cell viability, migration, and invasion in PCa cells. The expression of androgen receptor (AR), fatty acid synthase (FASN), and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) was substantially inhibited by AIF treatment in PCa cells. Furthermore, by inhibiting FASN and HMGCR expression, AIF decreased the amounts of intracellular fatty acids, cholesterols, and lipid droplets in PCa cells. Significantly, through coordinated targeting FASN- and HMGCR-regulated biosynthesis and the AR axis, AIF activated the caspase-associated apoptosis in PCa cells. These results collectively demonstrated for the first time the potential of AIF as a novel and attractive remedy and provided an alternative opportunity to cure PCa malignancy.

Feeding tannins to dairy cows in different seasons improves the oxidative status of blood plasma and the antioxidant capacity of cheese.

The aim of the present study was to assess the dietary supplementation of tannins to grazing dairy cows in 2 seasons characterized by a good quality pasture (spring) or a poor-quality pasture (summer). The effects of dietary tannins were assessed on plasma antioxidant status and cytokines profile and on the antioxidant properties of cheese and cheese in vitro digestates. Fourteen lactating dairy cows were divided into 2 homogeneous groups (n = 7): a control group (CON), and an experimental group (TAN) receiving 150 g/head per day of tannins supplementation. The experiment was performed twice, in spring and in summer. The animals were free to graze on spontaneous pasture (spring) or on dry stubble (summer). Blood was sampled at the beginning (d 0), at the midpoint (d 11), and at the end (d 22) of the trial. Individual cheese was produced before the beginning (d -1) and at the end (d 22) of the trial from the milk collected from each cow. On blood plasma, the reactive oxygen metabolites (ROM), biological antioxidant potential (BAP), nonesterified fatty acids quantification, and cytokines profile in terms of IL-10, IL-8, IL-1β, and IFN-γ were determined. Data on ROM demonstrated that tannins supplementation lowered oxidative stress both in spring and in summer. Accordingly, TAN diet increased BAP levels compared with the CON during summer trial. Thus, feeding tannins resulted in lower ratio between ROM and BAP (oxidative stress index) in both spring and summer. Cytokines' profile showed lower IL-1β values in TAN group at d 22 during spring season, with a concomitant higher IL-10 level, during summer season. Moreover, TAN group had a lower level of IFN-γ in plasma than CON group, both in spring and in summer. On cheese samples, the in vitro digestion was performed and on cheese and cheese digestates (gastric and intestinal digestate) the free radical scavenging antioxidant activity was evaluated. The intestinal digestate fraction registered the highest antioxidant activity compared with cheese and gastric digestate, in both spring and summer seasons. Furthermore, an improvement of the antioxidant property of cheese and cheese digestates was found. Present data demonstrated that tannins supplementation contributed to reduce the oxidative stress of lactating dairy cows and showed an increase of anti-inflammatory cytokines ratio.

A validated and optimized method for separation and quantification of total fatty acids by gas chromatography–ion trap mass spectrometry in human plasma

Fatty acids (FAs) are associated with many physiological functions of tissues, and their alteration has been linked with tissue-specific or systemic diseases. The current situation warrants us to have a sensitive and specific method for analysis of total FAs simultaneously from the biological fluid so that the risk prediction, diagnosis or prognosis of the disease can be made effectively. Because of greater sensitivity and resolution, a method of gas chromatography-ion trap mass spectrometry (GC–IT/MS) has been optimized and validated to quantify simultaneously 19 total FAs levels in plasma and compared with GC–triple quadrupole mass spectrometry. FAs have been transesterified by methanolic acetyl chloride to fatty acid methyl esters (FAMEs). A 65 min GC method separated all 19 FAMEs. The calibration curve had good linearity up to 313–922 μM with a correlation coefficient between 0.9882 and 0.9998. The LODs and LOQs of FAMEs were in the range of 0.63 to 9.55 and 2.12 to 31.8 μM, respectively. The method has recovery up to 144 %, stability at 4 °C for 48 h and one freeze–thaw cycle, and good intra-day and inter-day precision. The optimized method has been used to quantify plasma total FAs in type 2 diabetes mellitus patients with and without acute coronary syndrome. Though a significant difference has been found between IT/MS and triple quadrupole mass spectrometry, the GC–IT/MS can help to quantify total FAs in the clinical setting.

Quantitative Analysis and Structural Elucidation of Fatty Acids by Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tags and m-CPBA Epoxidation.

In this study, a novel analytical method was developed to investigate fatty acids (FAs) for relative quantification, carbon-carbon double-bond localization, and cis-/trans-geometry differentiation by isobaric multiplex labeling reagents for carbonyl-containing compound (SUGAR) tag conjugation and meta-chloroperoxybenzoic acid (m-CPBA) epoxidation. FAs are essential components of cells and have diverse functions in energy storage and as complex lipid constituents. It has been reported that FAs play different roles in various biological processes such as the functional development of the brain. The comprehensive characterization and quantification of FAs are crucial to further elucidate their biological roles. However, it is challenging to perform relative quantification and structural elucidation of FAs using integrated mass spectrometry (MS)-based methods. Recently, our group developed isobaric multiplex SUGAR tags for quantitative glycomics. Besides aldehyde/ketone groups on glycans, hydrazide groups also possess reactivity toward carboxylic acids on FAs. In this study, we extended SUGAR tag labeling with FAs for the quantitative analysis by liquid chromatography (LC)-MS/MS in the positive ion mode and applied this strategy for the comparative analysis of FAs hydrolyzed from oil samples. In addition, to comprehensively elucidate the structures of unsaturated FAs, epoxidation by m-CPBA was performed before SUGAR tag labeling to enable carbon-carbon double-bond localization. Moreover, the cis- and trans-geometries of carbon-carbon double bonds in multiple pairs of monounsaturated FAs could also be differentiated in higher-energy collisional dissociation (HCD)-MS/MS. This study developed a high-throughput comprehensive FA analysis platform, which could be widely applied and utilized in biological and clinical studies.

The effect of phenobarbital treatment on behavioral comorbidities and on the composition and function of the fecal microbiome in dogs with idiopathic epilepsy.

Phenobarbital (PB) is one of the most important antiseizure drugs (ASDs) to treat canine idiopathic epilepsy (IE). The effect of PB on the taxonomic changes in gastrointestinal microbiota (GIM) and their functions is less known, which may explain parts of its pharmacokinetic and pharmacodynamic properties, especially its antiseizure effect and drug responsiveness or drug resistance as well as its effect on behavioral comorbidities. Fecal samples of 12 dogs with IE were collected prior to the initiation of PB treatment and 90 days after oral PB treatment. The fecal samples were analyzed using shallow DNA shotgun sequencing, real-time polymerase chain reaction (qPCR)-based dysbiosis index (DI), and quantification of short-chain fatty acids (SCFAs). Behavioral comorbidities were evaluated using standardized online questionnaires, namely, a canine behavioral assessment and research questionnaire (cBARQ), canine cognitive dysfunction rating scale (CCDR), and an attention deficit hyperactivity disorder (ADHD) questionnaire. The results revealed no significant changes in alpha and beta diversity or in the DI, whereas only the abundance of Clostridiales was significantly decreased after PB treatment. Fecal SCFA measurement showed a significant increase in total fecal SCFA concentration and the concentrations of propionate and butyrate, while acetate concentrations revealed an upward trend after 90 days of treatment. In addition, the PB-Responder (PB-R) group had significantly higher butyrate levels compared to the PB-Non-Responder (PB-NR) group. Metagenomics of functional pathway genes demonstrated a significant increase in genes in trehalose biosynthesis, ribosomal synthesis, and gluconeogenesis, but a decrease in V-ATPase-related oxidative phosphorylation. For behavioral assessment, cBARQ analysis showed improvement in stranger-directed fear, non-social fear, and trainability, while there were no differences in ADHD-like behavior and canine cognitive dysfunction (CCD) scores after 90 days of PB treatment. While only very minor shifts in bacterial taxonomy were detected, the higher SCFA concentrations after PB treatment could be one of the key differences between PB-R and PB-NR. These results suggest functional changes in GIM in canine IE treatment.

Quaterization Derivatization with Bis(Pyridine) Iodine Tetrafluoroboride: High-Sensitivity Mass Spectrometric Analysis of Unsaturated Fatty Acids in Human Thyroid Tissues.

Accurate quantification of disease-related unsaturated fatty acids (UFAs) in biomedical samples plays an important role in clinical diagnosis. Here, we reported a quaterization derivatization-stable isotope labeling strategy for accurate quantitative analysis of UFAs by high-performance liquid chromatography-mass spectrometry. [d0]/[d10]-Bis(pyridine) iodine tetrafluoroboride ([d0]/[d10]-IPy2BF4) was employed as the carbon-carbon double bond derivatization reagent with high efficiency and high specificity, to introduce a charge tag on UFAs and avoid the interference of saturated fatty acids. After labeling, the detection sensitivity was significantly enhanced by up to three orders of magnitude compared to intact UFAs. The standard curves showed good linearity (R2 > 0.999) over a wide concentration range. This strategy was successfully applied to determine the content of 12 UFAs in human thyroid carcinoma and para-carcinoma tissues. A significant difference was found in the content of several UFAs between these two kinds of tissues (p < 0.05). These results indicated that the proposed strategy may be valuable for the discovery of abnormal UFA content in early clinical diagnosis.

Chia (Salvia hispanica) seeds degradation studied by fuzzy-c mean (FCM) and hyperspectral imaging and chemometrics - fatty acids quantification

Chia seeds are nutritious food because they have a high content of protein, polyunsaturated fatty acids (omega-3 and omega-6) and phenolic compounds. During storage, fatty acids are degraded, by oxidative and hydrolytic reactions, forming free fatty acids (FFA). In this work, we used Near Infrared Hyperspectral Imaging (NIR- HSI) and chemometrics to predict FFA acid value and fatty acids concentrations in chia seeds during storage. First, we explore the hyperspectral images by Fuzzy c-means (FCM), where it is possible to observe as chemical compounds are formed or degraded during storage. Second, PLSR models were developed to predict FFA value and fatty acids concentration. RPD values reached values higher then 2.0, indicating a good ability to estimate these chemical compounds, especially polyunsaturated fatty acids omega-3 and omega-6. Finally, NIR-hyperspectral imaging coupled with chemometrics allowed us to show the chemical degradation process of chia seeds during storage, mainly associated with polyunsaturated fatty acids degradation. Besides NIR-HSI showed to be a powerful technique to quantify the main fatty acids with high accuracy.

Invivo detection of triacylglycerols through Nile red staining and quantification of fatty acids in hyper lipid producer Nannochloropsis sp. cultured under adequate nitrogen and deficient nitrogen condition

The green alga Nannochloropsis sp. was grown in a medium containing adequate (1.5 g/L sodium nitrate) and absence of nitrogen (0 g/L sodium nitrate) for improving the triacylglycerols (TAGs) level and fatty acid content for sustainable biodiesel production. Initially, the biomass yield of Nannochloropsis sp. was determined from the day of inoculation till 18th day and it is observed that on 14th day higher biomass yield at about 1.4 g/L was obtained. The Nile red stained cells from nitrogen sufficient and nitrogen starvation conditions observed under confocal microscopy revealed that neutral lipids exist as oil bodies were higher in nitrogen starved condition and also, the TAG content was 13.31% dryweight in nitrogen starved cells than nitrogen amended (10.46%) Nannochloropsis cells. The extracted lipids subjected for FTIR study showed the presence of TAG functional group in both condition and similarly, the FAME obtained from both condition showed the vibration at 1743 cm−1 for ester functional group. The fatty acid composition of nitrogen deplete condition revealed oleic acid methyl ester (28.7%), Behenic acid methyl ester (14.2%), cis-11, 14, 17-Eicosatrienoic acid methyl ester (8.8%) as predominant fatty acids than nitrogen supplied condition. The TAG content and FAME composition reported in this study makes Nannochloropsis sp. as plausible candidate for pilot scale biodiesel production.

Method for absolute quantification of short chain fatty acids via reverse phase chromatography mass spectrometry.

Short chain fatty acids (SCFAs; including acetate, propionate, and butyrate) are an important class of biological molecules that play a major role in modulating host-microbiome interactions. Despite significant research into SCFA-mediated biological mechanisms, absolute quantification of these molecules in their native form by liquid chromatography mass spectrometry is challenging due to their relatively poor chromatographic properties. Herein, we introduce SQUAD, an isotope-based strategy for absolute quantification of SCFAs in complex biological samples. SQUAD uses aniline derivatization in conjunction with isotope dilution and analysis by reverse-phase liquid chromatography mass spectrometry. We show that SQUAD enables absolute quantification of biologically relevant SCFAs in complex biological samples with a lower limit of detection of 40 nM and a lower limit of quantification ranging from 160 nM to 310 nM. We observed an intra- and inter-day precision under 3% (relative standard deviation) and errors in intra- and inter-day accuracy under 10%. To demonstrate this quantification strategy, we analyzed SCFAs in the caecal contents of germ free versus conventionally raised specific pathogen free (SPF) mice. We showed that acetate was the most abundant SCFA in both types of mice and was present at 200-fold higher concentration in the SPF mice. We also illustrated the use of our quantification strategy in in vitro microbial cultures from five different species of bacteria grown in Mueller Hinton media. This study illustrates the diverse SCFA production rates across microbial taxa with acetate production serving as one of the key differentiating factors across the species. In summary, we introduce an isotope dilution strategy for absolute quantification of aniline-dativized SCFAs and illustrate the utility of this approach for microbiome research.