Purification and characterization of fatty acid-binding protein from human placenta

Purification of a cytosolic fatty acid-binding protein (FABP) from developing human placenta has been achieved, and its role in modulating the inhibition of human placental glucose-6-phosphate dehydrogenase (G6PD) by palmitoyl-CoA (PAL-CoA) has been studied. FABP was resolved into three peaks, viz. DE-I, DE-II and DE-III, by DEAE cellulose chromatography. DE-I was almost lipid-free. Presence of endogenous fatty acids in DE-II and DE-III was detected by thin layer chromatography (TLC). Fatty acids were the only detectable lipid component in these fractions. Gas liquid chromatography (GLC) analysis revealed that DE-II binds long chain saturated and unsaturated fatty acids nonspecifically, whereas DE-III is mainly an arachidonic acid carrier. Each of these fractions, viz. DE-I, DE-II and DE-III, has a molecular weight of 14,200 Daltons. Ouchterlony double immunodiffusion studies have confirmed the immunochemical identity of these three fractions of placental FABP. Separation in ion exchanger may be due to their different isoelectric points and varied types of binding affinities. Human placental G6PD was inhibited 50% by 0.03 mM PAL-CoA. The DE-II fraction of FABP enhanced the activity of G6PD in the absence of added PAL-CoA and protected against PAL-CoA inhibition of the enzyme. Such a modulating effect of FABP in this inhibition is attributable to binding of long chain acyl-CoA rather than to a direct effect of FABP on the enzyme itself.     

Oleic acid transfer from microsomes to egg lecithin liposomes: Participation of fatty acid binding protein

Oleic acid transfer from microsomes or mitochondria to egg lecithin liposomes was stimulated by fatty acid binding protein. By gel filtration, it could be demonstrated that this protein incorporates oleic acid into liposomes. Fatty acid binding protein transfer activity was higher using microsomes rather than mitochondria, which suggests a selective interaction with different kinds of membranes. Transfer of oleic acid by this soluble protein is greater than that of stearic acid. The results indicate that fatty acid binding protein may participate in the intracellular transport of fatty acids.     

Studies on lipid and fatty acid composition of human hepatoma tissue

Studies are reported on the composition of the lipids of human liver and hepatoma tissues from male adults. Liver tissues were obtained from individuals who died from causes other than liver disease or cancer. The hepatoma tissues were obtained from individuals shortly after they succumbed to cancer. The total lipid of each tissue was fractionated quantitatively by silicic acid column chromatography into neutral lipid, glycolipid, and phospholipid fractions. These fractions were analyzed by thin layer chromatography and converted to methyl esters for analysis of their constituent fatty acids by gas liquid chromatography. In comparison to liver tissue, the total amount of lipid in the hepatoma tissues was generally higher and more variable; the lipid of one hepatoma was ca. 92% of the dry wt of the tissue. The greater lipid content of the hepatoma tissues was due to the high percentage of neutral lipid. Except for one specimen, there was ca. the same amount of glycolipid in the hepatoma as in the liver tissues, but the composition of the glycolipid fraction of the hepatoma lipid differed considerably, particularly in the ganglioside fraction. The phospholipid fraction of hepatoma lipid was much lower than that of liver but exhibited only quantitative differences in composition. No glyceryl ether diesters and only traces of plasmalogens of phosphatidyl choline or phosphatidyl ethanolamine were detected in the liver and hepatoma lipids. The levels of monoenoic acids were higher and those of linoleic and polyunsaturated fatty acids lower in the hepatoma lipids. Positional isomers of trienoic acids not normally present in liver tissue were detected in hepatoma lipids. The abnormalities observed in lipid composition indicated interferences in the regulatory processes of lipid metabolism in human hepatoma similar to those observed in animals.     

Occurrence of octadecenoic fatty acid isomers from hydrogenated fats in human tissue lipid classes

The level oftrans-18∶1 isomers in several isolated lipid classes of human liver, heart, red blood cells and plasma was determined. Phospholipids contained substantially fewertrans-18∶1 isomers than triglycerides. The double bond distribution of thecis andtrans octadecenoate fraction of triglycerides and phosphatidylcholines from human liver and heart was determined. Whereas the double bond distribution of the triglycerides correlated closely with the pattern found in dietary hydrogenated vegetable oils, the phosphatidylcholine fraction showed evidence of selective incorporation or metabolism of specifictrans positional isomers. In general, isomers with double bonds near the methyl terminus were present at levels higher than expected from their relative abundance in the diet. Refinements in methodology needed to analyze octadecenoate double bond configuration and location in human tissues are presented.     

Geotrichum candidum NRRL Y-553 lipase: Purification,characterization and fatty acid specificity

Lipases fromGeotrichum candidum NRRL Y-553 are of interest because of their unique specificity forcis-9-unsaturated fatty acids relative to both stearic and palmitic acids. The lipases were partially purified by chromatography on Octyl Sepharose, AG MP-1 macroporous anion exchanger, and chromatofocusing resin. The preparation was found to contain multiple, glycosylated lipases varying slightly in pI (pI 4.88, 4.78, 4.65, 4.57 and 4.52) as judged by both activity and silver staining. The molecular mass determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 64 kilodaltons for the main species, with minor species of 60 and 57 kilodaltons present as well. The specificity of the crude lipases for hydrolysis of 4-methylumbelliferyl esters of oleicvs. palmitic acid was 20-to-1. The specificity of the purified, partially separated lipases was similar to that of the crude preparation. Thus the lipases could be used even in crude form for the hydrolysis and restructuring of triacylglycerols on a large scale.     

The fatty acid composition of human gliomas differs from that found in nonmalignant brain tissue

To compare the fatty acid composition of tumor tissue from glioma patients with that of normal brain tissue, tissue samples were obtained from 13 glioma patients and from 3 nonmalignant patients. Following lipid extraction, total fatty acid composition was measured using gas-liquid chromatography. Samples were further separated into phospholipids and neutral lipids. Representative samples were then separated into phospholipid classes by thin-layer chromatography and the fatty acid composition assayed. Levels of the polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA), were significantly reduced (P=0.029) in the glioma samples compared with normal brain samples; mean values were 4.8±2.9% and 9.2±1.0%, respectively. This reduction in glioma DHA content was also observed in terms of phospholipids (4.6±2.1% vs. 9.6±0.8%,P=0.002). The phosphatidylserine and phosphatidylethanolamine phospholipid classes were reduced in the glioma samples. Differences were also noted in the n-6 PUFA content between glioma and normal brain samples. The glioma content of the n-6 PUFA linoleic acid was significantly greater (P<0.05) than that observed in the control samples in terms of total lipids. Thus, the fatty acid composition of human gliomas differs from that found in nonmalignant brain tissue.     

Site-specific differences in the fatty acid composition of human adipose tissue

The fatty acid composition of triacylglycerols from fifteen distinct adipose depots taken from each of seven adult male human subjects was compared. Oleic, palmitic, linoleic, stearic, myristic, palmitoleic and vaccenic acids accounted for more than 90% of the triacylglycerol fatty acids in all sites from all subjects; a number of other fatty acids were also identified and quantified. There were large differences in theaverage fatty acid composition between individual subjects. There were no site-specific differences in the proportions of myristic (3.8–4.7% of triacylglycerol fatty acids), palmitic (23–29%), linoleic (6.7–9.8%) or vaccenic (4.1–4.7%) acids or in the proportions of any of the less abundant fatty acids. There were some significant site-specific differences in the proportions of palmitoleic, oleic and stearic acids. The calf depot contained more palmitoleic acid (6.41±1.09%) than the trapezius (3.12±0.55%), perirenal (3.59±0.50%) and mesenteric (3.70±0.43%) depots, more oleic acid (42.13±1.27%) than the trapezius (36.03±2.18%), perirenal (36.50±1.56%) and breast (37.13±1.55%) depots and less stearic acid (5.18±0.89%) than the trapezius (8.57±0.97%), perirenal (8.49±0.75%), mesenteric (7.87±0.42%), breast (8.02±0.75%) and clavicular (8.34±0.78%) depots. The buttock depot contained less stearic acid (6.06±0.65%) than the perirenal, mesenteric and clavicular depots, while the anterior thigh depot contained less stearic acid (6.07±0.70%) than the perirenal depot. These findings indicate that, while most human adipose depots differ little in fatty acid composition, some sites, in particular the calf, perirenal, trapezius and mesenteric depots, have site-specific properties.     

Purification of cyclic fatty acid esters: a GC-MS study

Gas chromatographic analysis of cyclic monomeric concentrates and fractions from argentation chromatography on packed columns containing SE-30, OV-25 and Apiezon L stationary phases yielded incompletely separated peaks representing the various isomers present in the mixture. Somewhat better separation was achieved using a 6 ft×1/8 in. column packed with 15% EGS on Chromosorb W. This column, when coupled to a mass spectrometer, yielded information concerning the composition of each of the isomeric components. Comparable results were obtained using a 50 ft×0.02 in. S.C.O.T. column with DEGS stationary phase and a 150 ft×0.01 in. capillary column coated with Apiezon L. While argentation thin layer chromatography proved useful, an argentation column method using silicic acid coated with 10% AgNO₃ proved more efficient for larger scale preparations. Elution of the column with 2% diethyl ether in petroleum ether yielded material essentially free of conjugated linolenate. A comparison of the behavior upon argentation thin layer chromatography of conjugated methyl linolenate, methyl linoleate and cyclic monomer esters indicated that these esters migrated to the same relative position as methyl oleate. Presented at the AOCS Meeting, Los Angeles, April 1972. Abstracted in part from the dissertation to be submitted to the University of Illinois Graduate College in partial fulfillment of the requirements for the Ph.D. degree.     

Phospholipid synthesis by chick retinal microsomes: Fatty acid preference and effect of fatty acid binding protein

The acylation of 1-palmitoyl-sn-glycerophosphocholine (1-16∶0-GPC) or 1-palmitoyl-sn-glycerophosphoethanolamine (1-16∶0-GPE) was measured using the microsomal fraction prepared from retinas of 14–15-day-old chick embryos. Rates of incorporation of exogenously supplied fatty acids into diacyl-GPC were generally 5–7 times greater than into diacyl-GPE. Substrate preferences for incorporation into diacyl-GPC and diacyl-GPE were, respectively, 18∶2>18∶3=20∶5>20∶4>18∶1>22∶6=18∶0 and 18∶2>22∶6≽18∶3=18∶0≽20∶4=18∶1>20∶5. The apparent selectivities were not consistent with the reported fatty acid compositions of these lipid classes. The addition of partially purified fatty acid binding protein (FABP) to the reaction had no effect either on overall rates of incorporation or on the substrate preference. When fatty acyl-CoA substrates were used, rates of incorporation of the 18∶0 derivative were much higher than with the fatty acid, while rates with other fatty acyl-CoA were similar to those with the respective fatty acid. Substrate preferences for CoA derivatives incorporated into diacyl-GPC were: 18∶0>20∶4>18∶2≽22∶6, and into diacyl-GPE: 20∶4=22∶6>18∶0>18∶2. Polyunsaturated fatty acyl CoA (PUFA-CoA) were thus favored for incorporation into diacyl-GPE, and to a lesser extent into diacyl-GPC, a result that is consistent with composition data. When purified FABP was added to the reactions, there was an increase in the incorporation of 18∶0-CoA and a decrease or no change in the incorporation of PUFA-CoA. The deacylation/reacylation cycle thus appears to play a role in the modification of phospholipid composition. The data are not consistent, however, with a role for FABP in directing PUFA toward membrane lipid synthesis.     

Systematic management and analysis of fatty acid data from multiple tissue samples

A systematic approach has been developed for the collection and analysis of gas chromatographic (GC) data from multiple fatty acid profiles. The approach was applied to a series of polar and nonpolar tissue lipids generated in animal feeding studies to allow a comparison of mean fatty acid profiles as a function of either dietary regimen or tissue location. The magnitude of the studies, sufficiently large to minimize error from animal variabilities, mandated the use of computer assistance. Nevertheless, manual input was essential due to the complexity of the GC patterns, and was invoked for peak assignment and report editing. The approach discussed here allowed for the consolidation and statistical analysis of data from over 30,000 GC peaks, and generated results in both tabular and graphic formats. It should be extendable to other chromatographic studies of lipid components. Agricultural Research Service, U.S. Department of Agriculture, 600 E. Mermaid Lane.     

Purification and characterization of recombinant human erythropoietin from milk of transgenic pigs

BACKGROUND: Human erythropoietin (hEPO), a hydrophobic acidic glycoprotein responsible for the regulation of red blood cell production in mammals, is used for the treatment of anemia. In general, the purification of transgenic animal‐derived therapeutic proteins is not easy due to their low titer concentrations and abundant contaminant proteins. For the first time, here the purification and characterization of rhEPO from the milk of transgenic pigs are described. RESULTS: The rhEPO was purified by heparin chromatography, reverse‐phase chromatography, and gel filtration chromatography, resulting in a 16.5% yield and > 98% purity. The rhEPO purified from the milk of transgenic pigs contained less acidic isoforms and was underglycosylated in contrast to CHO‐derived rhEPO. Cell proliferation of the F‐36/EPO‐dependent cell line was proportional to the dose of transgenic pig‐derived rhEPO. CONCLUSION: Transgenic pig‐derived rhEPO with high purity was achieved after three‐step chromatography following two‐step precipitation. The transgenic pig‐derived rhEPO was demonstrated to have comparable potency with CHO‐derived rhEPO. Transgenic pig‐derived rhEPO may not be therapeutically feasible because of different glycosylation, and thus further studies are required to elucidate the effect of this aberrant glycosylation on the biological activity and stability in vivo. Copyright © 2008 Society of Chemical Industry     

De novo fatty acid synthesis and fatty acid elongation catalyzed by subcellular fractions from hog and human aorta

De novo synthesis and mitochondrial elongation of fatty acids have been demonstrated in subcellular fractions from hog and human aorta. Microsomal fatty acid elongation has been shown in hog aorta. The activity catalyzing the formation of fatty acids from acetyl and malonyl CoA was associated with a high molecular weight complex in the 6×10⁶g×min supernatant fraction. The principal product was palmitic acid. Some myristic and stearic acids were also formed. One elongation system was associated with protein which sedimented between 4500 g×min and 150,000 g×min. It used acetyl CoA but not malonyl CoA, and NADH was the preferred reducing agent. Radioactivity from acetyl CoA was incorporated into many fatty acids. In hog aorta a second elongation system was found associated with protein which sedimented at 6×10⁶ g×min. It used malonyl CoA preferentially as substrate and either NADH or NADPH as reducing agent.     

Purification of polyunsaturated fatty acid esters from tuna oil with supercritical fluid chromatography

The technical and economic feasibility of producing docosahexaenoic acid (DHA)- and eicosapentaenoic acid (EPA)-ethyl ester concentrates from transesterified tuna oil using supercritical fluid chromatography (SFC) was studied. A systematic experimental procedure was used to find the optimal values for process parameters and the maximal production rate. DHA ester concentrates up to 95 wt% purity were obtained in one chromatographic step with SFC, using CO₂ as the mobile phase at 65°C and 145 bar and octadecyl silane-type reversed-phase silica as the stationary phase. DHA ester, 0.85 g/(kg stationary phase · h) and 0.23 g EPA ester/(kg stationary phase · h) can be simutaneously produced at the respective purities of 90 and 50 wt%. The process for producing 1,000 kg DHA concentrate and 410 kg EPA concentrate per year requires 160 kg stationary phase and 2.6 tons/h carbon dioxide eluant recycle. The SFC operating cost is U.S. $550/kg DHA and EPA ethyl ester concentrate.     

脂肪酸淀粉酯的合成与结构表征

脂肪酸淀粉酯是一种新型的非离子型表面活性剂,具有广泛的应用价值.以天然的淀粉和大豆油为原料,通过化学法进行酯化改性,制得混合脂肪酸淀粉酯.研究了基本的合成路线,并用FT-IR和UV对其结构进行表征.产物乳化性能比较优越.     

Purification and characterization of antioxidative peptides from protein hydrolysate of lecithin-free egg yolk

The protein extracted from lecithin-free egg yolk, normally discarded by lecithin processing plants, was hydrolyzed with the aid of Alcalase, a commercial enzyme. The hydrolysate was separated through a series of ultrafiltration membranes with molecular weight cutoffs of 10, 5, and 1 kDa; and three types of permeates including 10 K (permeate from 10 kDa), 5 K (permeate from 5 kDa), and 1 K (permeate from 1 kDa) were obtained. The antioxidative efficacy of hydrolysates so obtained was investigated and compared with α-tocopherol. Furthermore, two different peptides showing strong antioxidative activity were isolated from the hydrolysates by using consecutive chromatographic methods including ion exchange chromatography on a SP-Sephadex C-25 column, gel filtration on a Sephadex G-25 column, and high-performance liquid chromatography on an octadecylsilane column. The purity of the peptides was identified using capillary electrophoresis. The isolated peptides were composed of 10 and 15 amino acid residues, and both contained a leucine residue at their N-terminal positions.     

Intestinal and liver fatty acid binding proteins differentially affect fatty acid uptake and esterification in L-cells

Differential effects of intestinal (I-FABP) or liver (L-FABP) fatty acid binding proteins on fatty acid uptake and esterification were examined using transfected mouse L-cell fibroblasts. L-FABP, but not I-FABP, expression increased the initial rate and extent ofcis-parinaric acid uptake by 50 and 29%, respectively, compared to control cells. I-FABP and L-FABP expression preferentially increased [³H]-oleic acid incorporation into triacylglycerols by 5.5-fold and 3.8-fold, respectively. While both L-FABP and I-FABP increasedesterification of [³H]-oleic acid into ethanolamine glycerophospholipids, these proteins had opposite effect on esterification into choline glycerophospholipids. These data show for the first time that distinct FABP differentially affect both fatty acid uptake and intracellular esterification.     

Lipid binding in mitochondria from testes of normal and essential fatty acid deficient rats

Essential fatty acid (EFA) deficiency in rat causes severe degeneration of spermatogenic tissue. Previously it was shown that the distribution of lipid classes changes very little during tissue degeneration. However it is well known that the fatty acid spectrum in lipids from testicular tissue is altered drastically during EFA deficiency. The molecular binding of lipids in membrane structures might be altered when a larger amount of ω9-acids is present in the various lipid classes in testes of EFA-deficient rats. In the present studies comparison was made of the binding of lipids in testicular mitochondrial membranes from rats fed a fat-free diet or a diet containing 6% peanut oil for 26 weeks. Isolated mitochondria were coated on glass beads, then dried and packed into a column, whereafter the membrane lipids were eluted with solvents with increasing dielectric constants. The differences between the binding of lipid classes in supplemented and EFA-deficient rats were not pronounced, but a tendency to a weaker binding in the EFA-deficient rats was observed. However for both groups the various extracts showed marked differences in the distribution of lipid classes concurrent with the change of the eluent. This indicates a different kind of binding in the membrane, not only for different lipid classes, but also within a special lipid class. Thus both phosphatidylcholines (PC) and phosphatidylethanolamines (PE) were found in extracts with quite different dielectric constants. The fatty acid composition of PC and PE in the major fractions eluted with chloroform and ethanol, respectively, was essentially the same. This indicates that the successive release of phospholipids (PL) in these two fractions was not based on fatty acid solubility properties but on variable binding in the membrane structure. The introduction of ω9-polyenoic fatty acids instead of ω6-polyenoic fatty acids in the PL of mitochondria membranes from EFA-deficient rats seems to be the only deviation in the lipid pattern of EFA-supplemented and EFA-deficient animals, and might therefore be responsible for the symptons of EFA deficiency. Presented in part at the AOCS-ISF World Congress, Chicago, September, 1970.     

Bisoxathiolane sulfides from a dioxoene fatty acid

A quantitative preparation of bisoxathiolane from 9,12-dioxo-trans-10-octadecenoic acid is discussed. The reagents used are β-mercaptoethanol and BF₃₋ etherate in acetic acid. The structure of the product was established with the help of elemental analysis, infrared, nuclear magnetic resonance and mass spectroscopy data.     

Individual phospholipid contents and their fatty acid compositions in early normal human embryonic lung tissue

The concentrations of the individual phospholipids and their fatty acid compositions were determined in lung tissue obtained from 10–11 and 14–15 week pregancies. In this early stage of pregnancy, the amounts and fatty acid compositions of the individual phospholipids did not change substantially. Our human embryonic lung tissue results were compared with the corresponding data for infants and adults.     

Dietary fat and the fatty acid composition of tissue lipids

Some characteristics of the fatty acid composition of animal tissue lipids are described and the origins of tissue fatty acids are discussed briefly. The effect of dietary fat on composition of tissue lipids is discussed. Types of dietary fatty acids for which experimental work is described include polyunsaturated fatty acids, short-chain fatty acids, fatty acids with chain length greater than C₁₈,trans unsaturated fatty acids, fatty acids with conjugated double bonds, acetylenic fatty acids, branched-chain fatty acids and oxygenated fatty acids. The individuality of fatty acids is discussed in relation to their roles as components of tissue lipids.