Application of high-performance liquid chromatography of plasma fatty acids as their phenacyl esters to evaluate splanchnic and renal fatty acid balance in vivo

Plasma fatty acids from renal and hepatic veins, and arterialized hand vein obtained in 20 subjects before and after insulin infusion were separated by reversed-phase high-performance liquid chromatography following phenacyl esterification. Separation and quantification over the range 1.0–100 nmol per injection of nine fatty acids was achieved within 60 min using [²H₃₁]palmitic acid as internal standard. Analytical recoveries were greater than 90% and the intra- and inter-assay coefficients of variation were less than 2.5 and 4.0%, respectively. Following insulin infusion, net splanchnic uptake of total fatty acids decreased from 3.0±0.3 to 1.0±0.1 μmol/kg min (p<0.01), whereas net renal balance remained neutral (−0.04±0.04 vs. −0.06±0.03 μmol/kg min, p=N.S.). Individual fatty acid balance varied from a low of 0.012±0.005 (myristic acid) to a high of 0.95±0.08 (oleic acid) μmol/kg min across the splanchnic tissues and from 0.005±0.002 (stearic acid) to 0.21±0.1 (oleic acid) μmol/kg min across the kidney. There is a substantial diversity in changes in plasma concentration and regional balance of individual fatty acid during short-term fasting and hyperinsulinemia. This method is simple, accurate, and can be applied to assess individual fatty acid metabolism in vivo.     

Impaired plasma fatty acid oxidation in extremely obese women

Skeletal muscle from extremely obese individuals exhibits decreased lipid oxidation compared with muscle from lean controls. It is unknown whether this effect is observed in vivo or whether the phenotype is preserved after massive weight loss. The objective of this study was to compare free fatty acid (FFA) oxidation during rest and exercise in female subjects who were either lean [n = 7; body mass index (BMI) = 22.6 +/- 2.2 kg/m(2)] or extremely obese (n = 10; BMI = 40.8 +/- 5.4 kg/m(2)) or postgastric bypass patients who had lost >45 kg (weight reduced) (n = 6; BMI = 33.7 +/- 9.9 kg/m(2)) with the use of tracer ([(13)C]palmitate and [(14)C]acetate) methodology and indirect calorimetry. The lean group oxidized significantly more plasma FFA, as measured by percent fatty acid uptake oxidized, than the extremely obese or weight-reduced group during rest (66.6 +/- 14.9 vs. 41.5 +/- 16.4 vs. 39.9 +/- 15.3%) and exercise (86.3 +/- 11.9 vs. 56.3 +/- 22.1 vs. 57.3 +/- 20.3%, respectively). BMI significantly correlated with percent uptake oxidized during both rest (r = -0.455) and exercise (r = -0.459). In conclusion, extremely obese women and weight-reduced women both possess inherent defects in plasma FFA oxidation, which may play a role in massive weight gain and associated comorbidities.     

Comparative study of fatty acid composition in human and monkey aorta

The fatty acid composition of thoracic and abdominal aortic intima and media of normal human subjects and rhesus monkeys has been studied. Significantly higher values of unsaturated fatty acids as compared to saturated fatty acids have been noted in the intima of monkey as compared to man. The fatty acid profile of the aortic wall in these two species has provided a probable biochemical basis for the lesser incidence of atherosclerosis in macaques.     

Effect of long-term salinity on cellular antioxidants,compatible solute and fatty acid profile of Sweet Annie (Artemisia annua L.)

Impact of long-term salinity and subsequent oxidative stress was studied on cellular antioxidants, proline accumulation and lipid profile of Artemisia annua L. (Sweet Annie or Qinghao) which yields artemisinin (Qinghaosu), effective against cerebral malaria-causing strains of Plasmodium falciparum. Under salinity (0.0–160 mM NaCl), in A. annua, proline accumulation, contents of ascorbate and glutathione and activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and catalase (CAT) increased, but the contents of reduced forms of glutathione (GSH) and ascorbate declined. The fatty-acid profiling revealed a major salinity-induced shift towards long-chain and mono-saturated fatty acids. Myristic acid (14:0), palmitoleic acid (16:1), linoleic acid (18:2) and erucic acid (22:1) increased by 141%, 186%, 34% and 908%, respectively, in comparison with the control. Contents of oleic acid (18:1), linolenic acid (18:3), arachidonic acid (22:0) and lignoceric acid (24:0) decreased by 50%, 17%, 44% and 78%, respectively. Thus, in A. annua, salinity declines ascorbate and GSH contents. However, increased levels of proline and total glutathione (GSH + GSSG), and activities of antioxidant enzymes might provide a certain level of tolerance. Modification in fatty-acid composition might be a membrane adaptation to long-term salinity and oxidative stress.     

Biosynthesis of gamma-linolenic acid and beta-carotene by Zygomycetes fungi

Due to increasing demand for natural sources of both polyunsaturated fatty acids (PUFAs) and beta-carotene, 28 Zygomycetes fungal soil isolates were screened for their potential to synthesize these biologically active compounds. Although all fungi produced C₁₈ PUFAs, only nine strains also formed beta-carotene. Although Actinomucor elegans CCF 3218 was the best producer of gamma-linolenic acid (GLA) (251 mg/L), Umbelopsis isabellina CCF 2412 was found to be the most valuable fungus because of the dual production of GLA (217 mg/L) and beta-carotene (40.7 mg/L). The calculated ratio of formed PUFAs provided new insight into activities of individual fatty acid desaturases involved in biosynthetic pathways for various types of PUFAs. The maximal activity of delta-9 desaturase was accompanied by high accumulation of storage lipids in fungal cells. On the other hand, maximal activity of delta-15 desaturase was found in strains synthesizing low amounts of oleic acid due to diminished delta-9 desaturase. Activities of delta-6 desaturase showed competition for fatty acids engaged in n3, n6, and n9 biosynthetic pathways. Such knowledge about fatty acid desaturase activities provides new challenges for the regulation of biotechnological production of PUFAs by Zygomycetes fungi.     

Glucose stimulates a decrease of the fatty acid saturation degree in Acinetobacter calcoaceticus

The fatty acid composition of Acinetobacter calcoaceticus 69-V was determined under various growth conditions. Saturated, unsaturated, and hydroxy fatty acids with chain lengths of 12–18 carbon atoms predominated in the fatty acid profile. With acetate or propanol as growth substrates, the ratio of saturated to unsaturated fatty acids varied with changes in the temperature. This was the only adaptive mechanism detected that compensated for the physical effects of temperature alterations on the cell membranes. The fatty acid composition of A. calcoaceticus grown at 40 °C had a saturation degree of approximately 50%; after growth at 20 °C it was approximately 35%. In the presence of a carbon and energy source, A. calcoaceticus was able to respond to temperature reductions under oxic conditions regardless of whether fatty acid biosynthesis was inhibited or not. This suggests an aerobic mechanism of fatty acid biosynthesis and the involvement of a fatty acid desaturase system. Addition of the non-growth substrate, glucose, helped the organism to adapt to lower temperature. The molecular mechanism of the aid is not really understood. The oxidation of glucose could provide the desaturase either with electrons directly via a pyrrolo-quinoline-quinone-linked glucose dehydrogenase or with NADH after fatty acid degradation has been initiated by ATP generated by the oxidation of glucose. Received: 19 June 1998 / Accepted: 28 December 1998     

Influence of ethanol on fatty acid composition of phospholipids in cultured neurons

Animals chronically exposed to ethanol show changes in neural membrane lipids which may underlie the development of tolerance and physical dependence. The object of this study was to investigate changes in the fatty acid composition of neuronal phospholipids cultured in the presence of ethanol (55 or 110 mM) for periods up to 7 days. Decreases were observed in the percentage of individual and total saturated fatty acids, while the double bond index: total saturated fatty acid ratio, increased. These changes do not support the hypothesis that neural membrane lipid composition changes to counteract the fluidizing action of ethanol.     

Selective screening for fatty acid oxidation disorders by tandem mass spectrometry: difficulties in practical discrimination

In a selective screening for fatty acid oxidation disorders by tandem mass spectrometry, we tested the diagnostic ratios and acylcarnitine concentrations in sera or blood spots, which were reported to be specific to very long-chain acyl CoA dehydrogenase deficiency, carnitine palmitoyltransferase I deficiency, and carnitine palmitoyltransferase II deficiency. While the acylcarnitine profiles in the majority of these patients were typical in the respective disorders, some overlapping of the indices was observed between these patients and the infants, who showed symptoms mainly related to hypoglycemia but did not have the disorders mentioned above. Although the diagnostic ratio of tetradecenoylcarnitine to dodecanoylcarnitine for very long-chain acyl CoA dehydrogenase deficiency seemed to minimize the overlapping in this study, additional measures including careful assessment of clinical data and enzyme assays may be necessary for the diagnosis in atypical cases.     

Synthesis and antimycobacterial activity of isoniazid derivatives from renewable fatty acids

This work describes the synthesis of a series of fatty acid hydrazide derivatives of isoniazid (INH). The compounds were tested against Mycobacterium tuberculosis H37Rv (ATCC 27294) as well as INH-resistant (ATCC 35822 and 1896 HF) and rifampicin-resistant (ATCC 35338) M. tuberculosis strains. The fatty acid derivatives of INH showed high antimycobacterial potency against the studied strains, which is desirable for a pharmaceutical compound, suggesting that the increased lipophilicity of isoniazid plays an important role in its antimycobacterial activity.     

A genome-wide association study of n-3 and n-6 plasma fatty acids in a Singaporean Chinese population

Polyunsaturated fatty acids (PUFAs) have a major impact on human health. Recent genome-wide association studies (GWAS) have identified several genetic loci that are associated with plasma levels of n-3 and n-6 PUFAs in primarily subjects of European ancestry. However, the relevance of these findings has not been evaluated extensively in other ethnic groups. The primary aim of this study was to evaluate for genetic loci associated with n-3 and n-6 PUFAs and to validate the role of recently identified index loci using data from a Singaporean Chinese population. Using a GWAS approach, we evaluated associations with plasma concentrations of three n-3 PUFAs [alphalinolenic acid (ALA), eicosapentaenoic acid and docosahexaenoic acid], four n-6 PUFAs [linoleic acid (LA), gammalinolenic acid, dihomogammalinolenic acid (DGLA) and arachidonic acid], and estimates of delta-5 desaturase and delta-6 desaturase activities among the participants (N = 1361) of the Singaporean Chinese Health Study. Our results reveal robust genome-wide associations (p value <5 × 10⁻⁸) with ALA, all four n-6 PUFAs, and delta-6 desaturase activity at the FADS1/FADS2 locus. We further replicated the associations between common index variants at the NTAN1/PDXDC1 locus and n-6 PUFAs LA and DGLA, and between the JMJD1C locus and n-6 PUFA LA (p value between 0.0490 and 9.88 × 10⁻⁴). These associations were independent of dietary intake of PUFAs. In aggregate, we show that genetic loci that influence plasma concentrations of n-3 and n-6 PUFAs are shared across different ethnic groups.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-015-0502-2) contains supplementary material, which is available to authorized users.     

Standardizing methylation method during phospholipid fatty acid analysis to profile soil microbial communities

Phospholipid fatty acid (PLFA) as biomarkers, is widely used to profile microbial communities in environmental samples. However, PLFA extraction and derivatization protocols are not standardized and have widely varied among published studies. Specifically investigators have used either HCl/MeOH or KOH/MeOH or both for the methylation step of PLFA analysis, without justification or research to support either one. It seems likely that each method could have very different outcomes and conclusions for PLFA based studies. Therefore, the objective of this study was to determine the effect of catalyst type for methylation on detecting PLFAs and implications for interpreting microbial profiling in soil. Fatty acid samples extracted from soils obtained from a wetland, an intermittently flooded site, and an adjacent upland site were subjected to HCl/MeOH or KOH/MeOH catalyzed methylation procedures during PLFA analyses. The methylation method using HCl/MeOH resulted in significantly higher concentrations of most PLFAs than the KOH/MeOH method. Another important outcome was that fatty acids with a methyl group (18:1ω,7c 11Me, TBSA 10Me 18:0, 10Me 18:0, 17:0 10Me and 16:0 10Me being an actinomycetes biomarker) could not be detected by HCl/MeOH catalyzed methylation but were found in appreciable concentrations with KOH/MeOH method. From our results, because the HCl/MeOH method did not detect the fatty acids containing methyl groups that could strongly influence the microbial community profile, we recommend that the KOH/MeOH catalyzed transesterification method should become the standard procedure for PLFA profiling of soil microbial communities.     

Chemical evaluation of fatty acid desaturases as drug targets in Trypanosoma cruzi

Four positional isomers of Thiastearate (TS) and Isoxyl (Thiocarlide) were assayed as fatty acid desaturase inhibitors in Trypanosoma cruzi epimastigotes. 9-TS did not exert a significant effect on growth of T. cruzi, nor on the fatty acid profile of the parasite cells. One hundred micromolars of 10-TS totally inhibited growth, with an effective concentration for 50% growth inhibition (EC₅₀) of 3.0 ± 0.2 μM. Growth inhibition was reverted by supplementing the culture media with oleate. The fatty acid profile of treated cells revealed that conversion of stearate to oleate and palmitate to palmitoleate were drastically reduced and, as a consequence, the total level of unsaturated fatty acids decreased from 60% to 32%. Isoxyl, a known inhibitor of stearoyl-CoA Δ9 desaturase in mycobacteria, had similar effects on T. cruzi growth (EC₅₀ 2.0 ± 0.3 μM) and fatty acid content, indicating that Δ9 desaturase was the target of both drugs. 12- and 13-TS were inhibitors of growth with EC₅₀ values of 50 ± 2 and 10 ± 3 μM, respectively, but oleate or linoleate were unable to revert the effect. Both drugs increased the percentage of oleate and palmitate in the cell membrane and drastically reduced the content of linoleate from 38% to 16% and 12%, respectively, which is in agreement with a specific inhibition of oleate Δ12 desaturase. The absence of corresponding enzyme activity in mammalian cells and the significant structural differences between trypanosome and mammalian Δ9 desaturases, together with our results, highlight these enzymes as promising targets for selective chemotherapeutic intervention.     

Adaptational changes in the lipids and fatty acid profile of the cell and thylakoid membrane of rice plants exposed to sunlight

Adaptational changes occurring in the lipids and fatty acids of the cell and the thylakoid membrane in response to high light treatment, was studied in 30 days old rice (Oryza sativa L. cv. Jyothi) plants grown under low (150–200 μmol m⁻² s⁻¹) or moderate (600–800 μmol m⁻² s⁻¹) light conditions. Results were compared with rice plants grown in high (1200–2200 μmol m⁻² s⁻¹) light conditions. Exposure of rice plants and isolated chloroplast to high light, resulted in an increase in the amount of malonaldehyde, indicating oxidation of membrane lipids. Qualitative and quantitative changes in the phosphoglycolipids and quantitative changes in neutral lipids were observed in rice plants grown under the different growth conditions. A few of the phosphoglycolipids and neutral lipids were present exclusively in plants grown at low or moderate or high light, indicating requirement of different type of lipid composition of rice plants in response to their different growth irradiances. However, no significant quantitative changes were observed in the different saturated and unsaturated fatty acid groups of total lipids in low, moderate and high light grown rice plants, as a result of exposure to high light. No qualitative changes in the fatty acid composition due to difference in growth irradiance or high light treatment were seen. The changes observed in the phosphoglycolipids and neutral lipid composition of cell and thylakoid membrane of low, moderate and high light grown rice plants in response to high light, are probably the result of physiological changes in the rice plants, to sustain optimum structure and function of the cell and thylakoid membrane to maintain active physiological functions to endure high light conditions.     

Impact of physical exercise on visceral adipose tissue fatty acid profile and inflammation in response to a high-fat diet regimen

PurposeStudies associate specific fatty-acids (FA) with the pathophysiology of inflammation. We aimed to analyze the impact of exercise on adipose tissue FA profile in response to a high-fat diet (HFD) and to ascertain whether these exercise-induced changes in specific FA have repercussions on obesity-related inflammation.MethodsSprague-Dawley rats were assigned into sedentary, voluntary physical-activity (VPA) and endurance training (ET) groups fed a standard (S, 35kcal% fat) or high-fat (71kcal% fat) diets. VPA-animals had unrestricted access to wheel-running. After 9-wks, ET-animals engaged a running protocol for 8-wks, while maintained dietary treatments. The FA content in epididymal white-adipose tissue (eWAT) triglycerides was analyzed by gas-chromatography and the expression of inflammatory markers was determined using RT-qPCR, Western and slot blotting.ResultsEight-wks of ET reversed obesity-related anatomical features. HFD increased plasma tumor necrosis factor (TNF)-α content and eWAT monocyte chemoattractant protein (MCP)-1 protein expression. HFD decreased eWAT content of saturated FA and monounsaturated FA, while increased linoleic acid and prostaglandin E2 (PGE2) levels in eWAT. VPA decreased visceral adiposity, adipocyte size and MCP-1 in HFD-fed animals. The VPA and ET interventions diminished palmitoleic acid and increased linoleic acid in HFD-fed groups. Moreover, both interventions increased PGE2 levels in standard diet-fed groups and decreased in HFD. ET increased eWAT fatty acid desaturase 1 (FADS1) and elongase 5 (ELOVL5) protein content in both diet types. ET reduced eWAT inflammatory markers (TNF-α, IL-6), macrophage recruitment (MCP-1 and F4/80) and increased IL-10/TNF-α ratio in plasma and in eWAT in both diet types.ConclusionsExercise induced FA-specific changes independently of dietary FA composition, but only ET attenuated the inflammatory response in VAT of HFD-fed rats. Moreover, the exercise-induced FA changes did not correlate with the inflammatory response in VAT of rats submitted to HFD.     

Fatty acid induced remodeling within the human liver fatty acid-binding protein

We crystallized human liver fatty acid-binding protein (LFABP) in apo, holo, and intermediate states of palmitic acid engagement. Structural snapshots of fatty acid recognition, entry, and docking within LFABP support a heads-in mechanism for ligand entry. Apo-LFABP undergoes structural remodeling, where the first palmitate ingress creates the atomic environment for placement of the second palmitate. These new mechanistic insights will facilitate development of pharmacological agents against LFABP.     

Effect of high versus low doses of fat and vitamin A dietary supplementation on fatty acid composition of phospholipids in mice

Dietary fat and vitamin A provide important precursors for potent bioactive ligands of nuclear hormone receptors, which regulate various enzymes involved in lipid homeostasis, metabolism and inflammation. We determined the effects of dietary fat and dietary vitamin A on hepatic expression of two fatty acid metabolizing enzymes, elongase 6 (ELOVL6) and stearoyl-coenzyme A desaturase 1 (SCD1) and the concentration of saturated fatty acids (SAFA) and monounsaturated fatty acid (MUFA) of phospholipids in serum and liver. Mice (n = 6) were fed 4 weeks with diets containing 2, 5 and 25 % of fat or vitamin A (0, 2,500 and 326,500 RE/kg as retinyl palmitate). MUFAs and SAFAs were measured using GC and ESI–MS/MS. Hepatic expression of metabolizing enzymes was determined using QRT-PCR. ELOVL6 was significantly down-regulated in response to a high-fat diet (p < 0.001) and significantly up-regulated in response to low-fat diet (p < 0.05). SCD1 expression was significantly lower in high- versus low-fat diet (p < 0.05). The vitamin A content in the diet did not influence the hepatic expression of both enzymes. In plasma, the amounts of MUFAs bound to phospholipids significantly decreased in response to a high-fat diet and increased after a low-fat diet. This tendency was also observed in the liver for various phospholipids sub-classes. In summary, this study shows that fat content in the diet has a stronger impact than the content of vitamin A on hepatic gene expression of SCD1 and ELOVL6 and thereby on MUFA and SAFA concentrations in liver and plasma.     

Antimicrobial activity of fatty acids from fruits of Peucedanum cervaria and P. alsaticum

Plants of the genus Peucedanum have been used in traditional medicine for a long time to treat different diseases including infectious diseases. The hexane fruits extracts of Peucedanum cervaria and P. alsaticum were examined for antimicrobial activity and analyzed for their fatty acid content. Fatty acid composition of oils were analyzed by GC/FID in methyl ester form. Minimal inhibitory concentrations (MICs) of fatty acid fractions against twelve reference bacterial and yeast strains were performed by the twofold serial microdilution broth method. Fourteen fatty acids were identified. Oleic and linoleic acids were found to be dominant. The extracts from both plants examined exhibited inhibitory effects against Gram‐positive strains tested with different MIC values (0.25–2 mg/ml); however, extract from P. alsaticum possessed stronger antibacterial properties and a broader spectrum. The growth of Gram‐negative bacteria and Candida spp. strains was not inhibited even at the highest extract concentration used (MIC>4 mg/ml). Standard fatty acids exhibited inhibitory effects towards all bacterial and yeast strains used in this study; however, the majority of bacteria were more sensitive to linoleic than to oleic acid. These results revealed, for the first time, that hexane extracts obtained from fruits of P. alsaticum and P. cervaria possess moderate in vitro antibacterial activity against Gram‐positive bacteria including staphylococci. Linoleic and oleic acids appear to be the compounds responsible for this effect, and a synergistic antimicrobial effect between these two fatty acids was indicated.     

Changes in fatty acid composition in Pseudomonas putida and Pseudomonas stutzeri during naphthalene degradation

The effects of naphthalene on the whole cell-derived fatty acid composition of Pseudomonas putida and Pseudomonas stutzeri during naphthalene degradation were investigated. These strains differed in their abilities to degrade naphthalene and in 1,2-catechol dioxygenase activities. The cells of both strains reacted to the addition of naphthalene with an increase in the saturated/unsaturated ratio. The dynamic changes comprised also alterations in the percentage of hydroxy, cyclopropane and branched fatty acids. Upon the exposure of naphthalene, new fatty acids were detected.