Identification of fatty acid polymorphic modifications by infrared spectroscopy

IR spectra in the 7.5–25.0 μ region are shown to be unique for the individual long chain odd carbon numbered saturated fatty acids and for the monounsaturated even carbon numbered fatty acids, their isomers, and polymorphic modifications. IR spectra are presented which illustrate similarites and dissimilarities resulting from chain length differences and polymorphic variations for monounsaturated acids. X-ray crystal long spacings for the saturated odd carbon numbered acids are compared with literature values. X-ray crystal long spacings for monounsaturatedtrans acids are shown to be related to carbon chain length and independent of the location of unsaturation. Diffractograms and IR spectra of the 2 polymorphic modifications of elaidic acids are presented. IR spectra illustrating band differences forcis andtrans isomers are shown. ARS, USDA.     

Identification and characterization of fully deuterated fatty acids isolated fromScenedesmus obliquus cultured in deuterium oxide

The algaScenedesmus obliquus cultured in deuterated water, synthesized fully deuterated saturated and unsaturated long chain fatty acids. Their methyl esters were purified and their equivalent chain lengths were determined by gas liquid chromatography (GLC). They were characterized by mass spectroscopy, IR and near-IR spectroscopy, and NMR spectroscopy. Hexadeca-3,6-dienoic acid was identified. The fatty acid compositions of the total lipid and of individual lipid classes were measured. The melting point of methyl perdeuteriohexadecanoate was lower than that of its hydrogen counterpart. Methyl esters of perdeuterio fatty acids had shorter retention times in GLC chromatography on polar and nonpolar phases. Presented before ISF-AOCS Congress, Chicago, September 1970.     

Fatty acid composition of seed oils of some members of the meliaceae and combretaceae families

Seed oils of some members of the Meliceae (six) and Combretaceae (three) were analyzed for their fatty acid composition. In oils of members of both families palmitic acid was the most abundant saturated acid. Trace amounts of short chain (C12–C14) and long chain (C20–C22) saturated acids were detected in some members of the two families. Oleic acid was the most abundant unsaturated acids in the oils of four members of the Meliaceae. However, in the oils ofCedrella odorata andLovoa trichilloides, dienoic acid (C18:2) was the major unsaturated acid. Strikingly high levels of trienoic (C18:3) and monoenoic (C16:1) acids were detected in the seed oils ofC. odorata andEnthandrophragma angolense, respectively. Oleic acid also was the most abundant unsaturated acid in the Combretaceae. The nutritional value and industrial potentials of these oils are given.     

Microbial Conversion of Vegetable Oil to Rare Unsaturated Fatty Acids and Fatty Alcohols by an <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> Isolate

We isolated two new microorganisms capable of converting vegetable oil to several rare unsaturated fatty acids and rare unsaturated fatty alcohols from a soil sample. The strains were identified as belonging to the same genus and species, Aeromonas hydrophila. The rare unsaturated fatty acids and rare unsaturated fatty alcohols were accumulated as a wax ester form by the strains. Compared to other strains, the A. hydrophila isolates effectively decreased fatty acid chain lengths and converted rapeseed oil, which is rich in 9-C18:1 fatty acid, into rare fatty acids, such as 7-C16:1 fatty acid and 5-C14:1 fatty acid. Furthermore, the A. hydrophila isolates converted the resulting fatty acids to rare unsaturated fatty alcohols, such as 7-C16:1 fatty alcohol and 5-C14:1 fatty alcohol. The isolates also converted safflower oil, which is rich in 9,12-C18:2 fatty acid, to 7,10-C16:2 fatty acid, 5,8-C14:2 fatty acid, 9,12-C18:2 fatty alcohol, 7,10-C16:2 fatty alcohol, and 5,8-C14:2 fatty alcohol. 7,10,13-C16:3 fatty acid, 9,12,15-C18:3 fatty alcohol, and 7,10,13-C16:3 fatty alcohol were also converted from linseed oil, which is rich in 9,12,15-C18:3 fatty acid, by the A. hydrophila isolates. These fatty acids and fatty alcohols are rarely found in natural oils. Since decreasing fatty acid carbon chain lengths from the carboxyl end and reducing unsaturated fatty acids to unsaturated fatty alcohols are both difficult reactions to accomplish by chemical means, we suggest that these A. hydrophila isolates may facilitate introduction of new bioprocess for producing rare unsaturated fatty acids and rare unsaturated fatty alcohols, especially fatty alcohols harboring more than two double bonds.     

Different metabolism of saturated and unsaturated long chain plasma free fatty acids by intestinal mucosa of rats

During fat absorption, unsaturated long chain fatty acids are esterified at a higher rate than saturated fatty acids of similar chain length. This phenomenon has been attributed to differences in the binding affinity of fatty acids to a cytosolic fatty acid-binding protein. As intestinal mucosa utilizes plasma free fatty acids as well, we investigated whether long chainplasma free fatty acids of different degree of saturation are metabolized also at different rates.³H-Palmitic and¹⁴C-linoleic acid complexed to rat serum were injected rapidly into a tail vein of fasting rats. One, 2 and 4 min later there was no difference between³H and¹⁴C-radioactivity in intestinal mucosa, suggesting equal initial uptake of the two labeled fatty acids from plasma. Despite their equal uptake, the incorporation of the isotopes into ester lipids was significantly different, however: at 2 min, 53.1±3.9% of³H and 73.8±4.6% of¹⁴C were recovered in ester lipids. Phospholipids and triglycerides accounted for most of the mucosal³H and¹⁴C. At 4 min, a similar distribution of isotopes in intestinal mucosal metabolites was found. These data show that despite equal initial uptake by intestinal mucosa unsaturated long chain fatty acids taken up from plasma are esterified to a higher and oxidized to a lower extent than saturated plasma free fatty acids. Unsaturated plasma free fatty acids, therefore, may provide a more important source of fatty acids for endogenous intestinal lipoprotein lipids than saturated plasma free fatty acids. It is speculated that the fatty acid binding protein might be operative not only in the intracellular transport and metabolism of luminal fatty acids but of plasma free fatty acids as well.     

Cis and trans components of lipids: Analysis by 1H NMR and silver shift reagents

In this study, the methodology of shift reagents was exploited to distinguish cis and trans unsaturation in oils and fats. The differential binding of silver ions (in the form of AgFOD) to cis and trans double bonds allowed the separation of the allylic and olefinic proton signals in the ¹H NMR spectra of mixtures of cis and trans methyl esters of monoene aliphatic acids and unsaturated triacylglycerol mixtures at low frequency spectrometers (300 MHz). Careful integration of the appropriate proton resonances in the recorded quantitative ¹H NMR spectra afforded percentage concentrations in very good agreement with the actual values. This ¹H NMR methodology was validated by analyzing AOCS Laboratory Proficiency Program GC samples containing various percentages of saturated, cis‐mono unsaturated, and cis‐polyunsaturated fat as well as trans content. This fast and relatively low‐cost NMR methodology could be used on line for obtaining nutrition labeling compositional data (NLCD) required for fat‐containing food products. Attempts to differentiate lipid molecules with different degree of unsaturation and positional distribution of cis double bonds were unsuccessful.     

Synthesis of microbial elastomers based on soybean oil. Autoxidation kinetics,thermal and mechanical properties

Microbial bioelastomers prepared by the autoxidation of the unsaturated medium-long chain length co-poly-3-hydroxyalkanoate’s (mlcl-PHAs) based on soybean oily acids (Sy) have been reported. Pseudomonas oleovorans were grown on a series of the mixture of octanoic acid (OA) and Sy with the weight ratio of 20:80, 28:72 and 50:50 in order to obtain unsaturated mlcl-copolyesters coded PHO-Sy-2080, PHO-Sy-2872, and PHO-Sy-5050, respectively. The microorganism was also grown on the mixture of Sy and 10-undecenoic acid (UA) with the weight ratio of 50:50 to obtain unsaturated copolyester coded PHU-Sy-5050. The PHAs obtained were characterized by ¹H NMR and GC-MS techniques. Double bond contents of the unsaturated PHAs obtained were varying between 0.8 to 20 mol %. Autoxidation of the unsaturated copolyesters were carried out on exposure to air at room temperature in order to obtain new biomaterials whose mechanical strength was improved. Autoxidation kinetics, shelf life, mechanical and thermal properties of these biomaterials were evaluated.     

Composition of the neutral and phospholipid fractions from ginkgo nuts (Ginkgo biloba) and fatty acid composition of individual lipid classes

The purified lipid fraction (1.26% on the wet weight basis) from the nuts ofGinkgo biloba was found to be 90.6% neutral lipids, 7.5% polar lipids, and a very small amount of glycolipids. Main fatty acids in the triglyceride fraction were oleic and linoleic acids, and those in the phospholipid fraction were palmitic acid in addition to these unsaturated acids. The enzymic hydrolysis of the triglyceride and individual phospholipid fractions showed that only the triglyceride and phosphatidylcholine fractions contained relatively large amounts of unsaturated acids in their β-positions. The gas liquid chromatography-mass spectrometric analysis of the fatty acids of the steroid ester fraction indicated the presence of lignoceric, cerotic, montanic, and melissic acids as well as a lactone and compounds suspected to be phenolic acids containing long chain diols.     

Fractional vacuum distillation of herring oil methyl esters

Methyl esters of a Canadian Atlantic herring oil containing 62% monoethylenic fatty acids were subjected to batch fractional distillation under vacuum on a pilot plant scale, to study the feasibility of fractionating fatty acid esters of marine oils of low iodine value into monounsaturated fractions with increased commercial value for industrial chemical uses. A total of 64 methyl ester fractions were collected and analyzed by gas liquid chromatography. Recoveries of the major saturated and monounsaturated acids were close to 100%, and some fractions contained over 90% of the desired 22:1 long chain monounsaturated acids. The short chain polyunsaturated acids were recovered in good yields, but the long chain highly unsaturated acids were recovered in yields of 60% or less due to oxidative and thermal decomposition in the particular apparatus employed. If small amounts of unsaturated acids are acceptable, fractional distillation of low iodine value marine oils could inexpensively supply fractions with high concentrations of methyl esters of longer chain (C₂₀ and C₂₂) monounsaturated and shorter chain (C₁₄) saturated acid or (C₁₆) saturated-monounsaturated acid mixture.     

Fatty acid polymorph identification by infrared

IR spectra in the 7.5–25.0 μm region are shown to be unique for polymorphic modifications of long chain saturated fatty acids of even carbon number and also for the individual fatty acids. IR spectra are presented for the A-, B-, C- and E-forms of stearic acid and for the C-form of myristic, palmitic, arachidic and behenic acid and the differences discussed. X-ray determined crystal long spacings for the A-, B-, C- and E-forms of a series of homologous even carbon-numbered acids are presented and compared with literature values. The formation of the different polymorphic forms are found to be temperature-concentration related and affected little by polarity of the solvent. Wideline nuclear magnetic resonance second moment values are given for the polymorphic forms of stearic acid. Presented in part at the AOCS Meeting, Houston, May 1971. S. Market. Nutr. Res. Div., ARS, USDA.     

NMR chemical shift reagents in structural determination of lipid derivatives: II. Methyl petroselinate and methyl oleate

Whentris(1,1,1,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octaneodionato)europium (III)—Eu(fod)₃—forms a complex with a sufficiently basic functional group in a donor molecule, the change in the magnetic environment of protons near the coordination site causes their nuclear magnetic resonance (NMR) signals to shift to different positions. Consequently Eu(fod)₃ and other compounds that similarly affect NMR signals have been designated chemical shift reagents (csr). Because of their ability to shift proton signals, csr substantially increase the amount of structural information that can be obtained from NMR spectroscopy, frequently converting complicated splitting patterns into first-order spectra. Some generally useful experimental and interpretive csr techniques are described here using methyl petroselinate and methyl oleate as examples. Csr studies of methyl petroselinate reveal that the position of the double bond is at C-6, and that there is no chain substitution or branching before C-9. Csr studies of methyl oleate reveal that the position of the double bond is at or beyond C-9, and that there is no chain substitution or branching before C-6. Some suggestions are presented for expanding the amount of structural information that can be obtained by csr studies of unsaturated lipid derivatives. Presented in part at AOCS Meeting, Atlantic City, October, 1971.     

Lipid Adaptation of Shrimp Rimicaris exoculata in Hydrothermal Vent

The shrimp Rimicaris exoculata is the most abundant species in hydrothermal vents. Lipids, the component of membranes, play an important role in maintaining their function normally in such extreme environments. In order to understand the lipid adaptation of R. exoculata (HV shrimp) to hydrothermal vents, we compared its lipid profile with the coastal shrimp Litopenaeus vannamei (EZ shrimp) which lives in the euphotic zone, using ultra performance liquid chromatography electrospray ionization‐quadrupole time‐of‐flight mass spectrometry. As a result, the following lipid adaptation can be observed. (1) The proportion of 16:1 and 18:1, and non‐methylene interrupted fatty acid (48.9 and 6.2 %) in HV shrimp was higher than that in EZ shrimp (12.7 and 0 %). While highly‐unsaturated fatty acids were only present in the EZ shrimp. (2) Ceramide and sphingomyelin in the HV shrimp were enriched in d14:1 long chain base (96.5 and 100 %) and unsaturated fatty acids (67.1 and 57.7 %). While in the EZ shrimp, ceramide and sphingomyelin had the tendency to contain d16:1 long chain base (68.7 and 75 %) and saturated fatty acids (100 and 100 %). (3) Triacylglycerol content (1.998 ± 0.005 nmol/mg) in the HV shrimp was higher than that in the EZ shrimp (0.092 ± 0.005 nmol/mg). (4) Phosphatidylinositol and diacylglycerol containing highly‐unsaturated fatty acids were absent from the HV shrimp. (5) Lysophosphatidylcholine and lysophosphatidylethanolamine were rarely detected in the HV shrimp. A possible reason for such differences was the result of food resources and inhabiting environments. Therefore, these lipid classes mentioned above may be the biomarkers to compare the organisms from different environments, which will be benefit for the further exploitation of the hydrothermal environment.     

Quantification of Fatty Acids and their Regioisomeric Distribution in Triacylglycerols from Porcine and Bovine Sources Using 13C NMR Spectroscopy

The uptake of specific fatty acids in humans is dependent on their position on the glycerol backbone. There is a great interest in methods that can access this information fast and accurately. By way of high-resolution NMR, we have analyzed TAG extracted from pig and beef tissues and obtained quantitative data for the composition and regioisomeric distribution of all major unsaturated fatty acids usually found in these source materials, using a combination of manual integration and deconvolution of ¹³C NMR spectra. In addition, we have developed a method for determining composition and regioisomeric distribution of the two main saturated fatty acids found in pork (16:0, 18:0). The results are discussed in relation to species-specific genetic characteristics of fatty acid and TAG biosynthesis. The developed method could support decisions related to breeding for desired fatty acid profiles, and stimulate further methodology developments using high field NMR.     

The effect of feeding fish oils,vegetable oils and clofibrate on the ketogenesis from long chain fatty acids in hepatocytes

Groups of rats were fed diets containing 25% fish oil (FO), 25% soybean oil, 25% partially hydrogenated fish oil (PHFO), 25% partially hydrogenated soybean oil (PHSO), 25% partially hydrogenated coconut oil or 0.3% clofibrate for 3 wk. After the animals were fasted for 24 hr, hepatocytes were isolated and ketogenesis from added palmitate, linoleatecis andtrans, arachidonate and docosahexaenoate was measured. Ketogenesis after oil feeding was significantly stimulated (two-to threefold) only in cells from the PHFO-and PHSO-fed rats. The stimulation was most apparent with the long chain unsaturated fatty acids as substrates. These fatty acids were relatively poor ketone body precursors in control hepatocytes. Essential fatty acid deficiency did not seem to be the reason for this stimulation. Clofibrate also stimulated ketogenesis significantly (1.5- to 3-fold). The degree of stimulation increased with chain length and degree of unsaturation of the substrate. The activity of the enzyme 2,4-dienoyl-CoA reductase was also studied in the same groups. Its activity was stimulated about fourfold in the clofibrate-treated rats and to a lesser extent by the PHFO, PHSO and FO diets. The activity showed no correlation with the content of unsaturated fatty acids in the diet or their oxidation in isolated hepatocytes. The 2,4-dienoyl-CoA reductase, therefore, does not seem to be a regulatory enzyme in the metabolism of dietary polyunsaturated fatty acids. It is concluded that an induction of the peroxisomal β-oxidation system most likely is involved in the reported increases in ketogenesis from very long chain polyunsaturated fatty acids.     

<Superscript>1</Superscript>H- and <Superscript>13</Superscript>C-NMR Characterization of the Molecular Components of the Lipid Fraction of Pecorino Sardo Cheese

In this work the molecular fatty components of Pecorino Sardo Protected Designation of Origin (PS PDO) cheese were characterized through an exhaustive investigation of the ¹H- and ¹³C-NMR spectra of the extracted lipids. Several fatty acids (FA), such as long chain saturated, oleic, linoleic, linolenic, butyric, capric, caprylic, caproic, trans vaccenic, conjugated linoleic acid (cis9, trans11–18:2), and caproleic (9–10:1) were unambiguously detected. The positional isomery of some acyl groups in the glycerol backbone of triacylglycerols (TAG) was assessed. Furthermore, the NMR signals belonging to sn-1,2/2,3, sn-1,3 diacylglycerols (DAG), and free fatty acids (FFA) were analysed as a measure of lipolytic processes on cheese. Lastly, ¹H-NMR resonances of saturated aldehydes and hydroperoxides were detected, their very low intensity indicating that the lipid oxidation process can be considered to be of minor relevance in Pecorino Sardo cheese.     

Some physical,chemical, and biological properties of natural and synthetic unsaturated C18 acids

Series of octadecenoic (cis andtrans), octadecynoic, octadecadienoic (cis,cis andtrans,trans), and octadecadiynoic acids have been synthesized and employed in a comparative study of some physical (melting point, Raman spectra, NMR spectra, Ag⁺ thin layer chromatography, and gas liquid chromatography) and biological properties. The results show that the position of unsaturation has a considerable influence on the physical and biological properties of isomeric unsaturated acids. Several chemical reactions of linoleic acid and of the related oxygenated acids ricinoleic or vernolic (12,13-epoxyoleic) furnish cyclopropane compounds and 1,4- (or 1,5-) epoxides. The formation of these is rationalized in terms of neighboring group participation. The oxymercuration-demercuration reaction is the basis of a new method of examining acids with Δ3, Δ4, or Δ5 unsaturation. F.D. Gunstone received the 1973 Award in Lipid Chemistry at the AOCS 47th Annual Fall Meeting, Chicago, September 1973. This Award Address was presented at the Plenary Session.     

Profiling and Imaging of Phospholipids in Brains of Abcd1‐Deficient Mice

ABCD1 is a gene responsible for X‐linked adrenoleukodystrophy (X‐ALD), and is critical for the transport of very long‐chain fatty acids (VLCFA) into peroxisomes and subsequent β‐oxidation. VLCFA‐containing lipids accumulate in X‐ALD patients, although the effect of ABCD1‐deficiency on each lipid species in the central nervous system has not been fully characterized. In this study, each phospholipid and lysophospholipid species in Abcd1‐deficient mice brains were profiled by liquid chromatography‐mass spectrometry. Among the phospholipid and lysophospholipid species that are significantly more enriched in Abcd1‐deficient mice brains, VLCFA were present in 75, 15, 5, 4, and 1 species of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, and lysophosphatidylethanolamine, respectively. Most VLCFA were incorporated at the sn‐1 position of phosphatidylcholine and phosphatidylethanolamine. Among the phospholipid species that are significantly less enriched in Abcd1‐deficient mice brains, odd‐numbered saturated or mono‐unsaturated fatty acyl moieties are contained in all phosphatidylcholine species. In addition, a number of phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine species contained highly unsaturated fatty acyl moieties. Intriguingly, 44:1 phosphatidylcholine with VLCFA was mainly distributed in the gray matter, such as the cortex, but not in the white matter in the cerebrum and cerebellum. These results show that ABCD1‐deficiency causes metabolic alternation of long‐chain fatty acids and VLCFA. Moreover, our results imply a molecular mechanism for the incorporation of saturated or monounsaturated VLCFA into the sn‐1 position of phospholipids, and also indicate that the distribution of phospholipids with VLCFA may correlate with the development of X‐ALD.     

Chemical and physical properties of isomeric glyceryl monoethers

Long-chain saturated and mono- and diunsaturated 1- and 2-glyceryl monoethers were synthesized by reacting 1,2-isopropylidene and 1.3-benzylidene glycerol potassium salts with alkyl halides in the preparation of the saturated monoethers, and with alkenyl-p-toluenesulfonates in the preparation of the unsaturated monoethers, followed by hydrolysis of the blocking groups with boric acid. The progress of the reaction was monitored by gasliquid chromatography (GLC) of the reaction mixture. The 2-glyceryl ethers, with two exceptions, had not been prepared previously. Normal propyl and 3-pentyl octadecyl ethers also were synthesized to aid in the interpretation of infrared (IR) and nuclear magnetic resonance (NMR) spectra. All the ethers prepared were purified by preparative thin-layer chromatography (TLC) and crystallization. Their purity was found to be greater than 95%, as determined by TLC and GLC, supported by NMR and IR spectra. The isomeric 1- and 2-glyceryl ethers were separated on Silica Gel G adsorbent layers impregnated with either sodium arsenite or boric acid and their TLC behavior interpreted, based on the polarity of the complexes formed. Melting point determinations indicated more than one polymorphic form. Comparison of IR and NMR spectra of the saturated and unsaturated isomeric glyceryl ethers, and various derivatives, demonstrated the applicability of these spectroscopy methods for characterization and structural determination, in addition to distinguishing between the two isomeric forms. Presented at the AOCS Meeting, Philadelphia, October 1966. An operating unit of the Oak Ridge Associated Universities, under contract with the US Atomic Energy Commission.     

Analysis of oxidized biodiesel by 1H‐NMR and effect of contact area with air

Biodiesel is continuously gaining attention and significance as an alternative diesel fuel. An important issue facing biodiesel is fuel stability upon exposure to air due to its content of unsaturated fatty acids. Numerous factors influence the oxidative stability of biodiesel, and several methods for its assessment have been developed. In the present work, a defined amount of biodiesel (methyl soyate) was heated in open beakers, with the only difference being the size of the beaker, i.e. the surface area of the biodiesel exposed to air. Biodiesel oxidized in this fashion was analyzed by ¹H‐NMR, kinematic viscosity and acid value. Acid values and kinematic viscosity increased with time and surface area. A previously developed ¹H‐NMR procedure was used to evaluate the unsaturation and “residual” fatty acid composition. The amounts of saturated fatty acids determined by this method increased, with monounsaturated and diunsaturated species increasing and then decreasing with time. After “flash” (3 h, 165 °C) oxidation, NMR shows the greatest effect on saturates and compounds with two double bonds, the former increasing and the latter decreasing. The double bond originally located at δ15 in 18:3 is largely retained, showing that other double bond positions in 18:3 are initially affected by oxidation. The methyl ester signal decreases, coinciding with the increase in acid value. An increasingly strong absorption was observed in the UV‐VIS spectra. Increasing surface area accelerated oxidation and affected fatty acid composition.