Formation of omega- and (omega-1)-hydroxyfatty acids and plausible formation of ketofatty acid from microsomal phospholipids

Rat liver microsomes labeled with spin-labeled phosphatidylcholine release the label into the aqueous phase during the aerobic incubation with NADPH (Biochem. Biophys. Res. Commun. (1979) 87, 300-307). To establish the chemical nature of the released moiety, microsomes were labeled with [14C]phosphatidylcholine. When the 14C-labeled microsomes were incubated with NADPH under aerobic conditions, a few percent of the radioactivity was liberated into the aqueous phase within 60 min. Thin-layer chromatographic analysis of the radioactive substance liberated showed the presence of hydroxylated fatty acids derived from the 2-position of glycerol moiety. About one-third of the fatty acids formed from [14C]phosphatidylcholine during the incubation were converted into hydroxy-derivatives. Gas chromatography/mass spectrometry analysis further confirmed an NADPH-dependent formation of 16-hydroxypalmitic acid, 15-hydroxypalmitic acid, and hydroxy-derivatives of other fatty acids from the phospholipids of the microsomal membrane. Evidence was also obtained indicating the formation of ketopalmitic acid.     

Prostaglandin and fatty acid omega- and (omega-1)-oxidation in rabbit lung. Acetylenic fatty acid mechanism-based inactivators as specific inhibitors

Terminal acetylenic fatty acid mechanism-based inhibitors (Ortiz de Montellano, P. R., and Reich, N. O. (1984) J. Biol. Chem. 259, 4136-4141) were used as probes in determining the substrate specificity of rabbit lung cytochrome P-450 isozymes of pregnant animals in both microsomes and reconstituted systems. Lung microsomal and reconstituted P-450 form 5-catalyzed lauric acid omega- and (omega-1)-hydroxylase activities were inhibited by a 12-carbon terminal acetylenic fatty acid, 11-dodecynoic acid (11-DDYA), and an 18-carbon terminal acetylenic fatty acid, 17-octadecynoic acid (17-ODYA). Rabbit lung microsomal lauric acid omega-hydroxylase activity was more sensitive to inhibition by 11-DDYA than was (omega-1)-hydroxylase activity. In reconstituted systems containing purified P-450 form 5, both omega- and (omega-1)-hydroxylation of lauric acid were inhibited in parallel when either 11-DDYA or 17-ODYA was used. These data suggest the presence of at least two P-450 isozymes in rabbit lung microsomes capable of lauric acid omega-hydroxylation. This is the first report indicating the multiplicity of lauric acid hydroxylases in lung microsomes. Lung microsomal prostaglandin omega-hydroxylation, mediated by the pregnancy-inducible P-450PG-omega (Williams, D. E., Hale, S. E., Okita, R. T., and Masters, B. S. S. (1984) J. Biol. Chem. 259, 14600-14608) was subject to inhibition by 17-ODYA only, whereas 11-DDYA acid was not an effective inhibitor of this hydroxylase. We have recently developed a new terminal acetylenic fatty acid, 12-hydroxy-16-heptadecynoic acid (12-HHDYA), that contains a hydroxyl group at the omega-6 position. We show that 12-HHDYA possesses a high degree of selectivity for the inactivation of rabbit lung microsomal prostaglandin omega-hydroxylase activity which cannot be obtained with the long chain acetylenic inhibitor, 17-ODYA. In addition, 12-HHDYA has no effect on lauric acid omega- or omega-1-hydroxylation or on benzphetamine N-demethylation. The development of this new terminal acetylenic fatty acid inhibitor provides us with a useful tool with which to study the physiological role of prostaglandin omega-hydroxylation in the rabbit lung during pregnancy.     

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.     

High pressure liquid chromatography solvent systems for studies of bile acid biosynthesis

Reversed phase high pressure liquid chromatography (HPLC) solvent systems have been developed for the separation of intermediates in the formation of bile acids and bile acid conjugates from cholesterol. Four different mobile phases (water-methanol, 10 mM acetate buffer (pH 4.37)-methanol, 30 mM trifluoroacetic acid (pH 2.9 with triethylamine)-methanol, and 50 mM potassium phosphate buffer (pH 7.0)-2-propanol) have been applied to obtain separation of all the main intermediates with use of the same reversed phase column (Zorbax ODS).     

High performance liquid chromatography of nucleotides. Major methods and their development

The separation of mono- and oligonucleotides possibilities by means of high performance ion-exchange, reversed-phase, so-called "ion-pair" and adsorption chromatography are studied. The influence of the eluent composition (solvent, salt) and pH on the retention, selectivity and resolution in reversed-phase and ion-exchange chromatography is investigated. The model of the hydrophobic-pair ion-exchange mechanism of ion-pair chromatography is considered. The conditions for analysis and preparative isolation of a desired component are optimized for selectivity, resolution and throughput. The methods for prediction of the optimal gradient elution program reasonable resolution at the desired retention time and for choosing the guard-column packing material are proposed. A design of the gradient for system and the version of slurry packing method for HPLC prolonged life-time columns are improved. The automatized analytical technique for determination of the oligonucleotide monomeric composition with two coupled microcolumns is described, that involves enzymatic digestion of an oligonucleotide followed by ion-exchange separation of the hydrolysate.     

Direct separation of α-hydroxy acid enantiomers by ligand exchange chromatography

Direct separation of several α-hydroxy acid racemic mixtures was performed by the aid of ligand exchange chromatography using L-hydroxyproline chemically bound to silica stationary phase and aqueous solutions of copper (II) sulphate as a mobile phase. The elution order of the D- and L-enantiomers of α-hydroxy acids is interpreted in terms of a modified Davankov's rule. Several aspects of the Davankov's model of selectand-Cu(II)-selector ternary complexes are discussed based on the theoretical calculations within the quantum mechanical semiempirical and density functional theories. Chirality 10:821–830, 1998. © 1998 Wiley-Liss, Inc.     

High performance liquid chromatography of prostaglandins: Biological applications

Two procedures are described for separation and purification of prostaglandins by high performance liquid chromatography. Both systems show excellent resolution of PGA₂, PGE₂ and PGF_2α. Peak definition on the micro-particle silicic acid system is particularly good with the PGs appearing in 2–3 ml of organic effluent. Studies on reproducibility showed that PGE₂ and PGF_2α could be recovered with a retention volume of 54.2±0.76 ml and 64±0.6 ml, respectively (n=7, mean ±SD) with good recovery. The column can be run in about one hour and can be regenerated indefinitely (>200 times). The degree of purification is compatible with analysis by gas chromatography-mass spectrometry. Examples showing the application of this chromatographic method to human seminal fluid, human renal tissue, platelet rich plasma and human urine samples indicate that it makes possible analysis of these samples even at low levels.     

Incomplete fatty acid oxidation. The production and epimerization of 3-hydroxy fatty acids.

3-Hydroxydicarboxylic acids are major urinary metabolites derived from fatty acid metabolism. These compounds are produced from the omega-oxidation of 3-hydroxy fatty acids. The production of the precursor 3-hydroxy fatty acids from incomplete beta-oxidation of fatty acids in rat liver mitochondria was investigated. Independent of the chain length or the concentration of fatty acid substrates, the accumulation of 3-hydroxyacyl intermediates was relatively constant at the concentration of 3-5 nmol/mg of mitochondrial protein. The extent of the incomplete oxidation was the same in Percoll gradient-purified mitochondria. Rotenone treatment increased the production of 3-hydroxy fatty acids. 3-Hydroxy fatty acids did not exist as pure L-enantiomer as expected from beta-oxidation. Instead, these metabolites were epimerized to a near racemic mixture of D- and L-isomers with a slightly dominant D-isomer (58 +/- 3%). By using deuterium-isotope labeling, the mechanism of epimerizartion was shown to be a rapid dehydration-rehydration through trans-2-enoyl-CoA. In addition, cis-3 and trans-3 fatty acids were produced; these metabolites were derived from the isomerization of trans-2-enoyl-CoA. Epimerase and isomerase were thought to be enzymes involved in the oxidation of unsaturated fatty acids. Current data have shown that the metabolism of these acids is actually through NADPH-dependent reduction pathways. The activities of epimerase and isomerase detected in rat liver mitochondria possibly function mainly in the metabolism of saturated fatty acids in a reverse role to the conventional concept.     

High pressure liquid chromatography and widebore capillary gas-liquid chromatography methods for quantification of acetoin and diacetyl from bacterial cultures

Gas liquid chromatography (GLC) and high pressure liquid chromatography (HPLC) methods were developed to monitor acetoin and diacetyl levels during Lactobacillus plantarum fermentations. Low acetoin concentyration in culture filtrates were detected at 192 nm using a Polypore H cation column protected by a reverse phase C-18 guard column and elutated with 0.009 N sulfuric acid. Diacetyl was not detected at concentrations < 1800ppm. GLC proved to be a sensitive method for diacetyl when a wide-bore capillary Supelcowax column was used to analyze culture headspaces or ether extracts. Acetoin was not detected in culture headspaces, but concentrations > 150 ppm could be quantified from ether extracts.     

Nutrition and inflammatory events: highly unsaturated fatty acids (omega-3 vs omega-6) in surgical injury.

Given the poor prognosis and high cost of care for patients with acute inflammatory responses (often leading to organ failure and/or allograft rejection), immunomodulation of this hyperresponse represents an important priority for research in nutritional medicine. The primary goal of nutritional support in inflammatory disease is to provide adequate energy, particularly through use of novel lipids (to alter eicosanoid pathway toward a more regulated inflammatory state), and protein to meet endogenous requirements for tissue repair IL-1 production, and restored cellular function, thus preventing secondary infection (52). Manipulation of macrophage eicosanoid production by use of omega-3 PUFA may reduce the cellular immune response (by competing with arachidonic acid, which produces inflammatory eicosanoids of the 2- and 4-series), whereas inclusion of MCT found in coconut oil may lower the arachidonic acid content of membrane phospholipids. As more data are obtained on the use of such tailored therapies in critically ill patients, a new generation of parenteral and enteral diets will be developed to reduce inflammation and immune dysfunction.     

Interference of retinoic acid binding to its binding protein by omega-6 fatty acids

Cellular retinoic acid-binding protein (CRABP) is the putative mediator of the biological effects of retinoic acid in the control of epithelial differentiation and tumorigenesis. Omega-6 fatty acids such as linoleic acid and arachidonic acid, precursors of prostaglandin synthesis, caused inhibition of retinoic acid binding to CRABP. These fatty acids, however, possessed lower affinity than retinoic acid for the binding protein. Omega-3 fatty acids, such as eicosapentaenoic acid and docosohexaenoic acid, did not cause such inhibition in the binding of retinoic acid. Whereas retinoic acid was a potent modulator of differentiation of F9 embryonal carcinoma cells, neither omega-3 nor omega-6 fatty acids showed any significant differentiation potential. Competition by omega-6 fatty acids with retinoic acid for CRABP may neutralize the binding protein-mediated biological functions of retinoic acid, and could thereby enhance tumor production.     

Isolation and characterization of a seed-specific isoform of microsomal omega-6 fatty acid desaturase gene (FAD2-1B) from soybean.

In plants, the endoplasmic reticulum (ER)-associated oleate desaturase (FAD2) is the key enzyme responsible for the production of linoleic acid in non-photosynthetic tissues. In this study, we report the characterization of a seed-specific isoform of microsomal omega-6 fatty acid desaturase gene (FAD2-1B) sharing high sequence similarity with FAD2-1 from soybean. Several potential promoter elements including seed-specific motifs are found in the 5'-flanking region of FAD2-1B gene. The ORF of FAD2-1B is 1161 bp long and encodes a protein of 387 amino acids. This deduced protein holds three histidine boxes and four putative membrane-spanning helices, and possesses a signal for endoplasmic reticulum retention at C-terminal. Yeast cells transformed with the plasmid construct containing soybean FAD2-1B accumulate an appreciable amount of linoleic acid (18:2), normally not present in wild-type yeast cells, indicating that the cloned gene encodes a functional FAD2 enzyme. Both semi-quantitative RT-PCR and in silico analysis show that FAD2-1B gene is specifically expressed in developing seeds of soybean.