bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and specificity of lipases fromGeotrichum candidum
Authors: Baillargeon Mary Welch
Published: November 2006

A crude, commercialGeotrichum candidum lipase (EC 3.1.1.3) preparation (Amano GC‐20) was purified by hydrophobic interaction chromatography on Octyl Sepharose. The purified enzyme is a microheterogeneous glycoprotein containing isozymes varying in molecular weight, pI and specificity. It consists of 64, 62 and 59 kDa species as determined by denaturing polyacrylamide gel electrophoresis. Five isozymes (pI 4.40, 4.47, 4.58, 4.67 and 4.72) are detected by isoelectric focusing using both silver and activity stains. Chromatofocusing was used to separate the isozymes according to pI. Although all the isozymes are specific for oleatevs stearate esters, one isozyme (pI 4.72) is also specific for oleatevs palmitate. The number of isozymes is reduced to two (pI 4.67 and 4.72) after carbohydrate removal using endoglycosidase F/N‐glycosidase. These isozymes may be products of two lipase genes.
Formation of 8,15‐dihydroxy eicosatetraenoic acidvia 15‐ and 12‐lipoxygenases in fish gill
Authors: German J. B. , Berger Ralf
Published: November 2006

Metabolites of arachidonic acid are important regulators of biological function in a variety of mammalian tissues. We have demonstrated similar lipoxygenase enzyme activities in fish gills and mammalian lungs suggesting that their metabolites may have matching functions. Fish gills were investigated for their ability to generate dioxygenated metabolites of polyunsaturated fatty acids. Fatty acids, including arachidonic acid, were incubated with crude tissue homogenates and polar metabolites were extracted, derivatized and analyzed by high performance liquid chromatography, gas chromatography and mass spectrometry. The major dihydroxy metabolite of arachidonic acid was characterized as 8(LR), 15(LS)‐dihydroxyeicosatetraenoic acid. This product was formed by the sequential action of the 15‐ and 12‐lipoxygenases in the tissue. The formation of the dihydroxyeicosatetraenoic acid by crude tissue homogenates was significantly enhanced by the addition of 1 mM reduced glutathione. The metabolism of other polyunsaturated fatty acids, including eicosapentaenoic acid and docosahexaenoic acid, to dihydroxy acids was consistent with their relative ability to serve as substrates for the initial 15‐lipoxygenase reaction.
The metabolism of 20‐ and 22‐carbon unsaturated acids in rat heart and myocytes as mediated by feeding fish oil
Authors: Mohammed B. Selma , Hagve Tor‐Arne , Sprecher Howard
Published: November 2006

When rats were fed 5% corn oil, the heart phospholipids contained large amounts of 22‐carbon (n−6) acids. When half of the corn oil was replaced with fish oil, the reduced level of arachidonate and 22‐carbon (n−6) acids in phospholipids was accompanied by increases in the levels of 22‐carbon (n−3) acids while only small amounts of 20∶5(n−3) were acylated. Heart myocytes readily took up and acylated [1‐14C]‐labeled 20∶4(n−6), 20∶5(n−3) and 22∶6(n−3) into phospholipids. The uptake and acylation of 20∶4(n−6) was greater than for 20∶5(n−3) but the intracellular labeling profiles were similar. Uptake and acylation of 22∶6(n−3) was somewhat lower. In addition the intracellular labeling profile differed in that more 22∶6(n−3) was incorporated into the ethanolamine‐containing phospholipids than when 20∶4(n−6) or 20∶5(n−3) were the substrates. Neither 20∶4(n−6) nor 20∶5(n−3) was chain elongated. When [3‐14C]‐labeled 22∶4(n−6) and 22∶5(n−3) were the substrates, it was not possible to detect radioactive 22∶5(n−6) or 22∶6(n−3). Both [3‐14]‐labeled substrates were acylated into phospholipids and retroconverted with the subsequent esterification of radioactive 20∶4(n−6) and 20∶5(n−3) into triglycerides and phospholipids. These studies show that cardiomyocytes lack the ability to make 22‐carbon acids from 20‐carbon precursors but they retroconvert 22‐carbon acids to 20‐carbon acids. The high levels of 22‐carbon polyunsaturated acids in total heart lipids thus cannot be attributed to the synthetic capacities of cardiomyocytes.
Separation of 20∶4n−6 and 20∶4n−7 by capillary gas‐liquid chromatography
Authors: Wolff Robert L. , Sebedio Jean‐Louis , Grandgirard Andre
Published: November 2006

The use of a capillary column coated with 100% cyanopropyl polysiloxane (CPTMSil 88) allows the separation of several fatty acids associated with fat deficiency. Starting with liver mitochondrial phospholipids of weanling rats fed a fat‐free diet, an unusual fatty acid was isolated, along with 20∶4n−6, by thin‐layer chromatography on AgNO3‐impregnated silica gel plates. After partial hydrazine reduction of these acids, the resulting monoenes were isolated and subjected to ozonolysis in BF3/methanol. The resulting monomethyl and dimethyl esters were identified by gas chromatography/mass spectrometry. Our data indicate that the unusual component corresponds to 20∶4n−7. Based on published biochemical and analytical studies and on our own chromatographic retention data, some of the other unusual fatty acids were tentatively identified as 18∶2n−7, 20∶2n−7 and 20∶3n−7. The CPTMSil 88 column appears to be a simple and useful tool for the separation of fatty acids of the palmitoleate series.
The effect of bleomycin on the uptake and incorporation of [14C] choline into phospholipids in hamster lung tissue slices
Authors: Hollinger Mannfred A. , Giri Shri N.
Published: November 2006

Intratracheal administration of the anticancer drug bleomycin to hamsters produced an increase in the uptake and incorporation of [14C] choline into phospholipids of lung slicesin vitro. The stimulatory effect is opposite to the results obtained previously using [14C] acetate and would appear to occur distal to cytidine diphosphocholine. Although alternate explanations are possible, the results are consistent with morphological evidence, published by others, indicating an increase in lung phospholipid following bleomycin treatment, and illustrate the significance of precursor selection when evaluating the effects of xenobiotics on phospholipid synthesis.
Phosphatidylcholine as substrate for human pancreatic phospholipase A2. Importance of the physical state of the substrate
Authors: Borgström Bengt
Published: November 2006

The long‐chain phosphatidylcholine/sodium cholate aqueous system as substrate for human pancreatic phospholipase A2 (PLA2) was investigated. At a constant phosphatidylcholine (PC) concentration of 8 mM, the enzyme activity increased with a decrease in cholate (C) concentration up to a PC/C ratio of approximately 0.8 and then rather abruptly decreased to lower values at a ratio above 1.5. At ratios between 0.8 and 1.5, an increasing lag phase in the PLA2 activity was seen, indicating a progressive decrease in substrate availability to the enzyme. Reaction mixtures with a PC/C ratio of up to 0.67 were optically clear solutions composed of mixed bile salt/PC micelles of increasing mixed micellar aggregate size. Ratios between 0.67 and 1.5 were characterized by an increase in turbidity (at 330 and 450 nm) due to increasing formation of vesicles or liposomes. Above a PC/C ratio of 1.5, a sharp increase in turbidity was seen due to increasing formation of bilayer structures other than vesicles. Pure vesicles obtained by dialysis of mixed micellar solutions were not hydrolyzed by the enzyme. Addition of bile salts reversed the inhibition which was accompanied by a decrease in turbidity. Phosphatidylcholine was preferred as substrate for human PLA2 when present in large mixed disc‐like bile salt micelles. Vesicular or other types of lamellar liquid‐crystalline phases of long‐chain phosphatidylcholine did not serve as substrate for PLA2.
Purification and properties of an extracellular lipase fromPythium ultimum
Authors: Mozaffar Zahid , Weete John D.
Published: November 2006

An extracellular triacylglycerol lipase (EC 3.1.1.3) fromPythium ultimum strain No. 144 was purified by ammonium sulfate precipitation, and by diethylaminoethyl Sepharose CL‐6B and Sephacryl S−200 chromatography. The purified enzyme preparation showed a prominent polypeptide band in polyacrylamide gel electrophoresis, associated with esterase activity according to activity staining. Molecular weight of the protein was estimated at 270 kD using gel filtration on Sephacryl S−200, and 68 kD by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis indicating that the enzyme may be a tetramer. The optimum pH and temperature for activity of the enzyme were 8.0 and 30°C, respectively. Activity was reduced by Co2+, Fe2+, Sn2+ and Mn2+ and stimulated by Ca2+, Mg2+, Na+, K+ and surfactants such as taurocholic acid, Triton X−100,n‐octyl glucoside,n‐dodecyl‐β‐D‐maltoside, 3‐[(3‐cholamidopropyl) dimethylammonio]‐1‐propanesulfonate(CHAPS), and 3‐[‐cholamidopropyl)dimethylammonio]‐2‐hydroxy‐1‐propanesulfonate. The apparent maximum specific activity was 42 μmole/min/mg in the absence of CHAPS and 77 μmole/min/mg in its presence. The reaction rate was progressively higher with increasing number of double bonds in the substrate, and the enzyme showed a preference for triacylglycerols containing fatty acids having thecis double bond configuration.
Purification and characterization of a fatty acid binding protein from human prostatic tissue
Authors: Chaudry Aziz A. , Dutta‐Roy Asim K.
Published: November 2006

Epidemiological studies suggest the existence of a strong relationship between the incidence of prostatic cancer and the intake of dietary lipids in humans. However, very little information is available on intracellular fatty acid metabolism in human prostatic tissue. The objective of this study was to identify and subsequently characterize a fatty acid binding protein of human prostatic tissue. A fatty acid binding protein (FABP) was purified and characterized from human prostatic tissue. The purified FABP had an apparent molecular mass of 15.0±1.0 kDa as averaged from three different methods, sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), gel filtration and amino acid analysis. The pI value of the protein was determined to be 6.8. Scatchard analysis of fatty acid binding to the purified FABP from malignant prostatic tissue showed a Kd value of 0.53±0.02 μM for arachidonic acid (n=5). The Kd values of FABP purified from benign prostatic tissue were 0.57±0.02 μM for oleic acid and 0.51±0.04 μM for arachidonic acid (n=5). Fatty acid analysis revealed that the level of endogenously bound arachidonic acid was about 2.5‐fold higher in FABP from malignant than from benign tissue. In addition, both malignant and benign tissues contained the same concentration of FABP. The concentrations of FABP in malignant and benign tissues were 19.2±1.8 and 21.4±2.1 μg per mg of total cytosolic protein, respectively. Characterization based on amino acid composition, isoelectric point and fluorescence with dansyl undecanoic acid suggests that the FABP may not be of the heart type, but is rather more closely related to the liver type. As malignant prostatic tissue produces more PGE2 compared to benign tissue, our data suggest that FABP may help enhancing the synthesis of the prostaglandin in malignant tissue by facilitating arachidonic acid transport.
Trans‐monoenoic and polyunsaturated fatty acids in phospholipids of aVibrio species of bacterium in relation to growth conditions
Authors: Henderson R. J. , Millar R. ‐M. , Sargent J. R. , Jostensen J. ‐P.
Published: November 2006

AVibrio species of bacterium known to contain the polyunsaturated fatty acid 20∶5n−3 was grown in both freshwater and seawater media at 5 and 20°C and examined for adaptive changes in lipid composition. Phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), together with a smaller proportion of nonesterified fatty acids (NEFA), comprised almost all the lipid under all growth conditions examined. Temperature had a more pronounced effect than the salinity of the medium on lipid composition. The proportion of PE in total lipid was always higher at 5 than at 20°C. Conversely, the proportion of NEFA was lower at 5 than 20°C whereas that of PG was not altered. The levels of saturated fatty acids in total lipid, PE and PG were all decreased by growth at 5°C. No differences were observed with respect to growth temperature in the levels ofcis 16∶1n−7, the principal monoenoic fatty acid in both PE and PG.Trans 16∶1n−7 was found to comprise 12.8–15.2% of fatty acids in PE and PG of bacteria grown at 5°C but only 4.4–8.5% of phospholipid fatty acids in bacteria cultured at 20°C. Regardless of medium composition, a reduction in growth temperature from 20 to 5°C also caused the proportions of 20∶5n−3 to increase from around 0.8 to 4.4% in PE and from around 4 to 20% in PG. The simultaneous occurrence oftrans 16∶1n−7 and 20∶5n−3 is unique to thisVibrio species of bacterium. The increased proportions of both these fatty acids with decreasing temperature suggest that they have a role in retailoring biomembrane phospholipids during temperature acclimation of the bacterium.
2‐Hydroxyhexadecanoic and 8,9,13‐trihydroxydocosanoic acid accumulation by yeasts treated with fumonisin B1
Authors: Kaneshiro Tsuneo , Vesonder Ronald F. , Peterson Robert E. , Bagby Marvin O.
Published: November 2006

Fumonisin B1 is a sphingolipid‐like compound that enhances the accumulation of yeast sphingolipids and 2‐hydroxy fatty acids. These lipids occur both as freely extractable and cell bound components in yeast fermentations. Both free and bound 2‐hydroxy fatty acids produced byPichia sydowiorum NRRL Y‐7130 were increased when fumonisin B1 (50 mg/L) was added to the usual growth medium containing yeast extract/malt extract/peptone/glucose. Fumonisin‐treated cultures contained 38 mg/L more 2‐hydroxyhexadecanoic and 15 mg/L more 2‐hydroxyoctadecanoic acids than did untreated cultures. By contrast, fumonisin inhibited the accumulation of free 8,9,13‐trihydroxydocosanoic acid inRhodotorula sp. YB‐2501 cultures, leading to 240 mg/L lower trihydroxy acid production than by untreated cultures.
Mechanism of decreased arachidonic acid in the renal cortex of rats with diabetes mellitus
Authors: Ramsammy Leslie S. , Haynes Brenda , Josepovitz Christine , Kaloyanides George J.
Published: November 2006

The purpose of this study was to investigate the roles of decreased synthesis and increased consumption in the depression of arachidonic acid levels in renal cortex and glomeruli of rats with streptozotocin‐induced diabetes mellitus. In diabetic rats, arachidonic acid was depressed 33.2% in renal cortex, 47.4% in liver and 66.1% in heart compared to values of control rats. Δ6 Desaturase activity was depressed in renal cortex, liver and heart of diabetic rats to 53.3, 55.5 and 63.7%, respectively, of control values. Δ5 Desaturase activity was also depressed 43.7, 55.5 and 47.6% in renal cortex, liver and heart of diabetic rats, respectively. In other rats the activities of five enzymes involved in the synthesis and esterification of arachidonic acid were measured in renal cortex and in isolated glomeruli. Both tissues from diabetic rats showed depressed activities of Δ5 and Δ6 desaturases, increased activities of long‐chain acyl‐CoA synthetase and 1‐acyl‐sn‐glycero‐3‐phosphocholine acyltransferase and no change in the activity of elongase as compared to those in control tissues. Malondialdehyde, an end product of lipid peroxidation, was lower in the renal cortex of diabetic rats than in control rats, whereas β‐oxidation of linoleic acid and arachidonic acid were similar in diabetic and in control rats. Basal and stimulated prostaglandin E2 synthesis were significantly higher in isolated glomeruli from diabetic rats compared to those in control rats. In isolated tubules, prostaglandin E2 synthesis was similarly low in both groups. From these data we conclude that the reduced level of arachidonic acid esterified in lipids of the kidney cortex is caused principally by depressed synthesis of arachidonic acid secondary to decreased activity of Δ5 and Δ6 desaturases. Increased consumption of arachidonic acid to support prostaglandin synthesis may have contributed to the depression of arachidonic acid in glomeruli but not in tubules.
Effect of high fat corn oil, olive oil and fish oil on phospholipid fatty acid composition in male F344 rats
Authors: Rao Chinthalapally V. , Zang Edith , Reddy Bandaru S.
Published: November 2006

Epidemiological and laboratory animal model studies have provided evidence that the effect of dietary fat on colon tumorigenesis depends on the amount of fat and its composition. Because of the importance of the composition of dietary fat and of tissue membrane fatty acid composition in tumor promotion, experiments were designed to investigate the relative effects of high fat diets rich in ω3, ω6 and ω9 fatty acids and colon carcinogen on the phospholipid fatty acid composition of liver, colon, small intestine, erythrocytes and blood plasma. At 6 wk of age, groups of animals were fed diets containing 5% corn oil (LFCO), 23.5% corn oil (HFCO), 23.5% olive oil (HFOO), and 20.5% fish oil plus 3% corn oil (HFFO). Two weeks later all the animals except the vehicle‐treated animals received azoxymethanes.c. once weekly for 2 wk at a dose rate of 15 mg/kg body weight. Animals were sacrificed 5 d later and liver, colon, small intestine and erythrocytes and blood plasma were analyzed for phospholipid fatty acids. The results indicate that the phospholipid fatty acid composition of liver, colon and small intestine of HFCO diet fed animals, were not significantly different from those fed the LFCO diet. The levels of palmitoleic acid and linoleic acid were increased in erythrocytes and blood plasma of the animals fed the HFCO diet compared to those fed the LFCO diet. Feeding the HFCO diet significantly increased the oleic acid content and decreased the linoleic acid and arachidonic acid levels in various organs when compared to the HFCO diet. Animals fed the HFFO diet showed a marked increase in eicosapentaenoic acid and docosahexaenoic acid and a decrease in linoleic acid and arachidonic acid levels as compared to those fed the HFCO diet. The results also indicate that carcinogen treatment had only a minimal effect on the phospholipid fatty acid composition.
Effects of dietary proteins on linoleic acid desaturation and membrane fluidity in rat liver microsomes
Authors: Koba Kazunori , Wakamatsu Kosaburo , Obata Keisuke , Sugano Michihiro
Published: November 2006

The effect of dietary protein, casein (CAS) and soybean protein (SOY), on linoleic acid desaturation in liver microsomes was studied in rats. The activity of Δ6 desaturase in total and rough endoplasmic reticula (ER and RER) was significantly higher in the CAS group than in the SOY group. In ER and smooth endoplasmic reticulum, the steady‐state fluorescence anisotropy of 1,6‐diphenyl‐1,3,5‐hexatriene, when incorporated into the membrane, was decreased in the SOY group and accompanied by a reduction in the cholesterol/phospholipid (CHOL/PL) ratio, consistent with an increase in membrane fluidity. In a separate study, the effect of varying dietary proteins, CAS, milk whey protein, egg albumin, SOY, potato protein and wheat gluten, on the relationship between the Δ6 desaturase activity and microsomal membrane fluidity was also examined. The results indicated that the dietary protein‐dependent change in the liver microsomal CHOL/PL ratio affected membrane fluidity, and subsequently the activity of Δ6 desaturase in liver microsomes. However, since dietary protein influenced the Δ6 desaturase activity in RER without influencing membrane fluidity, it is possible that some regulation might have taken place at the level of enzyme synthesis.
Sterols ofChaetoceros andSkeletonema
Authors: Tsitsa‐Tzardis E. , Patterson G. W. , Wikfors G. H. , Gladu P. K. , Harrison D.
Published: November 2006

Dietary sterol is required by the oyster for growth, and sterol is believed to be obtained primarily from dietary phytoplankton. Seven isolates ofChaetoceros and one ofSkeletonema, which are of potential use as oyster food, were analyzed for sterol composition using gas chromatography, high‐performance liquid chromatography and gas chromatography/mass spectrometry.Skeletonema and five isolates ofChaetoceros contained cholesterol as their major sterol. Two other isolates ofChaetoceros also contained cholesterol, but 24‐methylenecholesterol was the principal sterol. Cholesterol has rarely been reported as the major sterol from phytoplankton. In view of the widespread occurrence ofSkeletonema andChaetoceros in the marine environment, these algae could be an important source of the oyster's cholesterol.
Changes in fatty acid desaturation in hepatic and intestinal tissues induced by intestinal resection
Authors: Vázquez C. M. , Muriana F. J. G. , Ruiz‐Gutierrez V.
Published: November 2006

We have studied the activities of Δ9, Δ6 and Δ5 desaturases in rat liver and jejunum when the entero‐hepatic circulation was interrupted by either 50 or 75% intestinal resection. Desaturase activities in caecal mucosa were also determined in control and operated rats. Distal small bowel resection significantly altered the activities of desaturase enzymes in liver, jejunum and caecum. Thus, the Δ9 desaturase activity was lower in hepatic microsomes from operated animals, and this decrease was not related to the extent by which the intestine was removed. However, a significant increase in both Δ6 and Δ5 desaturases was found in these animals compared to shamoperated rats, the increase in both desaturases being higher after 75% than after 50% intestinal resection. The activities of Δ9 and Δ5 desaturases were significantly increased in jejunal mucosa of resected rats. The activity of Δ6 desaturase was increased only in 50% resected‐animals. An increase in Δ6 desaturase activity was observed in caecal mucosa after resection, together with a decrease in Δ9 desaturase and no change in Δ5 desaturase activities. Enzyme activities were highest in the liver relative to the activity in jejunal and caecal homogenates. In sham rats, the caecal mucosa showed higher Δ9 and Δ6 but lower Δ5 desaturase activities than in jejunal mucosa.
Specific accumulation of docosahexaenoic acid (22∶6n−3) in brain lipids during development of juvenile turbotScophthalmus maximus L.
Authors: Mourente Gabriel , Tocher Douglas R. , Sargent John R.
Published: November 2006

The changes in the lipid class and the fatty acid compositions of total lipids and individual glycerophospholipids which occur in brain during development of juvenile turbot (Scophthalmus maximus L.) were investigated. Fish were sampled during a 10‐week period immediately following weaning from a live feed to a pellet diet. During this period, brain dry weight increased over 6‐fold. The percentages of protein, cholesterol and galactolipids increased in brain during development, presumably reflecting increased membrane maturation and myelination processes. The percentages of docosahexaenoic acid (DHA; 22∶6n−3) were low at the beginning of the study period. However, DHA specifically accumulated in juvenile turbot brain during development. The percentages of DHA increased in total lipid, total diradyl glycerophosphocholine (GPC), total diradyl glycerophosphoethanolamine (GPE), phosphatidylserine (PS) and phosphatidylinositol (PI) reaching 26.1%, 25.8%, 40.8%, 47.1% and 17.9% of the total fatty acids, respectively, by the end of the 10‐week period. The percentages of other n−3 polyunsaturated fatty acids (PUFA) and n−6 PUFA generally decreased during this period, as did that of monoenes; the percentages of saturated fatty acids remained relatively constant. Non‐linear regression analysis showed that the increase in DHA in total lipid, GPC, GPE and PS fitted 1st order rate kinetics (plus offset) allowing maximum values for the percentages of DHA in each lipid class to be estimated.
Metabolic transformation of intracraneally injected [1‐14C]linoleic and [1‐14C]α‐linolenic acids in malnourished developing rats
Authors: De Tomas Maria Elena , Mercuri Osvaldo , Serres Cristina
Published: November 2006

The effect of a low protein diet during pregnancy and lactation on the fatty acid composition of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from brains of ten‐day‐old rats was studied. The results indicated that partial deprivation of protein during early development was associated with an increase in the fatty acids of the n−9 family in PC. The fatty acids of the linoleic acid series decreased in PE but were not modified in PC. These minor changes did not affect the double bond index values either in PC or in PE. The effect of protein depletion on thein vivo metabolic transformation of intracraneally injected [1‐14C]linoleic and [1‐14C]α‐linolenic acids was also studied. The percentage distribution of the labeled precursors and their derivatives among PC and PE differed from that of mass distribution. These results indicate that the direct uptake of polyunsaturated fatty acids from the blood and/or the low turnover rate of these acids incorporated into PC and PE might be involved in maintaining the fatty acid pattern of these brain lipids.
Effect of dietary fat on rat liver microsomal and mitochondrial/lysosomal dolichol, phospholipid and cholesterol
Authors: Jakobsson‐Borin Åsa , Tollbom Örjan , Dallner Gustav
Published: November 2006

The influence of different fat diets on liver phospholipid, cholesterol and dolichol was studied. Rats were separated into four groups and fed standard laboratory chow (control), a diet containing linolenic acid, a coconut oil diet, or a corn oil‐containing diet. After five weeks, microsomes and mitochondrial/lysosomal fractions were prepared from the liver, and lipid compositions were analyzed. No changes in phospholipid content were observed. In control animals, the fatty acid compositions of phosphatidylcholine and phosphatidylethanolamine in the two subfractions were similar. However, these two phospholipids showed different fatty acid patterns, which were altered independently upon dietary treatment. The dietary treatments resulted, in most cases, in decreased cholesterol and dolichol contents and, especially in microsomes, in a decreased level of esterification of both lipids. The fatty acid compositions of cholesteryl esters in the two subfractions showed significant differences and cholesterol was esterified to a large extent with linolenic acid when this fatty acid was supplied in the diet. The same dietary treatment exerted different effects on the cholesterol localized in the two different intracellular compartments. This difference was most pronounced in rats fed the corn oil‐containing diet; microsomal cholesteryl esters exhibited increased saturation, whereas cholesteryl esters in the mitochondrial/lysosomal fraction displayed decreased saturation. Dolichyl esters in the two cellular compartments had different fatty acyl compositions, with a considerably higher degree of saturation in microsomes. The various diets influenced the nature of the fatty acid moieties present in the isolated fractions and the effects on the two subfractions were opposite. The diet containing linolenic acid decreased the degree of saturation in microsomal dolichyl esters and increased the degree of saturation in the mitochondrial/lysosomal fraction. The results demonstrate that the fatty acid compositions of both dolichyl and cholesteryl esters display organelle specificity. Both the content of these lipids and their fatty acid compositions are greatly influenced by dietary conditions, and the esterification processes at different cellular locations exhibit independent regulation, regardless of the fatty acid content of the diet.
Formation of dityrosine and other fluorescent amino acids by reaction of amino acids with lipid hydroperoxides
Authors: Kikugawa Kiyomi , Kato Tetsuta , Hayasaka Akira
Published: November 2006

Formation of fluorescence by the reaction of various amino acids with lipid hydroperoxides,i.e., linoleic acid 13‐monohydroperoxide, methyl linoleate 13‐monohydroperoxide and phosphatidylcholine hydroperoxide, in the presence of methemoglobin was investigated. Two types of fluorescence were produced: fluorescent dityrosine (3,3′‐dityrosine) from tyrosine, and unidentified fluorophores with α‐ and ε‐amino groups of various amino acids. While the former was stable after treatment with borohydride, the latter fluorophores were readily destroyed. The rate of dityrosine formation was rapid, and the yield of dityrosine was dependent on the concentrations of tyrosine and the lipid hydroperoxides. Butylated hydroxytoluene and tocopherol inhibited the formation of dityrosine, but did not affect the formation of fluorophores on the amino groups. Dityrosine appears to be formed by radical reaction of the lipid hydroperoxides, while the other fluorophores seem to be created by nonradical mechanisms.
Changes in lipids of pigeon “milk” in the first week of its secretion
Authors: Shetty Sreerama , Hegde S. N.
Published: November 2006

Pigeon “milk” (PM) collected from the crop of 1‐ to 5‐day‐old squabs was analyzed to examine whether there were changes in lipid composition during the first week of secretion. The high PM fat content (9–11%) remained fairly constant in the first 5 days of secretion. The mean percentage of neutral lipids, glycolipids and phospholipids was 80, 12 and 8%, respectively. Unlike the content of neutral lipids, glycolipid and phospholipid levels increased significantly between day 1 and day 5 of secretion. Triglycerides, the major neutral lipids, decreased by 24% between day 1 and day 5, while free sterols, monoglycerides and hydrocarbons increased by 8%, 11% and 2.5%, respectively, during the same period; diglycerides and sterol esters, however, remained unchanged. The ratio of saturated to unsaturated fatty acids was 0.27 and it remained unchanged. Medium‐chain (C10, C12 and C14) and oddchain (C15 and C17) fatty acid contents were low. Fatty acids longer than C20 were absent. Palmitic acid, the major saturated fatty acid, increased by 42% from day 1 to day 5, whereas stearic acid decreased by 48% during the same period. Oleic acid, the predominant unsaturated fatty acid, also decreased from 51 to 45% between the first and fifth day of PM secretion. Polyunsaturated acids (18∶2, 18∶3 and 20∶4) accounted for 26% and 30% of the total fatty acids on day 1 and day 5, respectively. Although lipid changes in the crop of squabs prior to collection of samples cannot totally be ruled out, the nature of lipid changes is likely to reflect cellular breakdown that precedes PM secretion by parent pigeons.
Identification of natural diacylglycerols as the 3,5‐dinitrophenylurethanes by chiral phase liquid chromatography with mass spectrometry
Authors: Itabashi Y. , Marai L. , Kuksis A.
Published: November 2006

This study reports a facile identification of the molecular species of enantiomeric diacylglycerols by combining chiral phase high‐performance liquid chromatography with positive chemical ionization mass spectrometry. For this purpose the 3,5‐dinitrophenylurethane (DNPU) derivatives ofsn‐1,2(2,3)‐diacylglycerols are separated on an (R+)‐naphthylethylamine polymer column (25 cm × 0.46 cm ID) using an isocratic solvent system made up of hexane/dichloroethane/acetonitrile (85∶10∶5, by vol) or isooctane/tert‐butyl methyl ether/acetonitrile/isopropanol (80∶10∶5∶5, by vol). About 1% of the column effluent (1 mL/min) was admitted to a quadrupole mass spectrometer (Hewlett‐Packard, Palo Alto, CA)via direct liquid inlet interface, and positive chemical ionization spectra were recorded over the range of 200–900 mass units. The DNPU derivatives of diacylglycerols yield characteristic [M‐DNPU]+ and [RCO+74]+ ions for each diacylglycerol species from which the molecular weight and exact pairing of fatty acids can be unequivocally obtained. The characteristic ions appear to be generated in nearly correct mass proportions as indicated by preliminary quantitative comparisons.
Inhibition of theClostridium perfringens phospholipase C hydrolysis of a thiophosphate analog of lysophosphatidylcholine by micelle‐bound ammonium and sulfonium cations
Authors: Young Paul R. , Snyder William R. , McMahon Rosemary F.
Published: November 2006

Cetyltrimethylammonium andn‐octadecyldimethylsulfonium bromides inhibit theClostridium perfringens phospholipase C‐catalyzed hydrolysis of 1‐S‐phosphocholine‐2‐O‐hexadecanoyl‐1‐mercapto‐2‐ethanol (1) at pH 7.5, 37°C, μ=0.15 with KCl. Mixed micelles containing 1 and either inhibitor are substrates for the enzyme and the fraction of activity remaining is a monotonic, but nonlinear function of the mole fraction of inhibitor. Simple saturation kinetics are observed as the concentration of 1 is increased in mixed micelles containing a constant mole fraction of inhibitor. Inhibition constants for cetyltrimethylammonium andn‐octadecyldimethylsulfonium bromides are 0.66±0.04 and 0.25±0.02 mM, respectively. The data suggest that the significant inhibition previously observed for soluble alkyldisulfonium salts (K50 for dodecamethylene‐bis(dimethylsulfonium) bromide, 27μM) is dependent upon bifunctional cationic interactions rather than hydrophobic binding.
An aldehyde‐containing galactolipid from the red algaGracilariopsis lemaneiformis
Authors: Jiang Zhi D. , Gerwick William H.
Published: November 2006

An unprecedented aldehyde‐containing digalactosyldi‐acylglycerol (DGDAG) has been isolated from the red algaGracilariopsis lemaneiformis and structurally defined. Its structure was determined as 1‐O‐[5‐hydroxy‐12‐oxododeca‐6(E), 8(E), 10(E)‐trienoyl]‐2‐O‐palmitoyl‐3‐O‐[‐β‐D‐galactopyranosyl‐6,1‐α‐D‐galactopyranosyl]‐glycerol by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and degradation to simple fragments. The identification of this aldehyde‐containing DGDAG fromG. lemaneiformis, in addition to our previous findings of unusual galactolipids, further indicates that this alga has unique lipoxygenase capacities.
The hypotriglyceridemic effect of eicosapentaenoic acid in rats is reflected in increased mitochondrial fatty acid oxidation followed by diminished lipogenesis
Authors: Willumsen Nina , Skorve Jon , Hexeberg Sofie , Rustan Arild C. , Berge Rolf K.
Published: November 2006

The effect of eicosapentaenoic acid (EPA) on fatty acid oxidation and on key enzymes of triglyceride metabolism and lipogenesis was investigated in the liver of rats. Repeated administration of EPA to normolipidemic rats resulted in a time‐dependent decrease in plasma triglycerides, phospholipids and cholesterol. The triglyceride‐lowering effect was observed after one day of feeding whereas lowering of plasma cholesterol and phospholipids was observed after five days of treatment. The triglyceride content of liver was reduced after two‐day treatment. At that time, increased mitochondrial fatty acid oxidation occurred whereas mitochondrial and microsomal glycerophosphate acyltransferase was inhibited. The phosphatidate phosphohydrolase activity was unchanged. Adenosine triphosphate:citrate lyase, acetyl‐CoA carboxylase, fatty acid synthetase and glucose‐6‐phosphate dehydrogenase were inhibited during the 15 d of EPA treatment whereas peroxisomal β‐oxidation was increased. At one day of feeding, however, when the hypotriglyceridemic effect was established, the lipogenic enzyme activities were reduced to the same extent in palmitic acid‐treated animals as in EPA‐treated rats. In cultured rat hepatocytes, the oxidation of [14C]palmitic acid to carbon dioxide and acid‐soluble products was stimulated in the presence of EPA. These results suggest that the instant hypolipidemia in rats given EPA could be explained at least in part by a sudden increase in mitochondrial fatty acid oxidation, thereby reducing the availability of fatty acids for lipid synthesis in the liver for export,e.g., in the form of very low density lipoproteins, even before EPA induced peroxisomal fatty acid oxidation, reduced triglyceride biosynthesis and diminished lipogenesis.
Plasma clerance and hepatic utilization of stearic, myristic and linoleic acids introducedvia chylomicrons in rats
Authors: Wang Shu , Koo Sung I.
Published: November 2006

The primary objective of the present study was to compare the rates of plasma clearance and hepatic utilization of stearic (18∶0), myristic (14∶0) and linoleic (18∶2) acids, as introducedvia chylomicrons. Lymph chylomicrons were specifically labeledin vivo with [14C]stearic and (SA), [14C]myristic acid (MA), or [14C]linoleic acid (LA) by infusing donor rats intraduodenally with the labeled fatty acids in a lipid emulsion. Following intravenous injection of recipient rats with the labeled chylomicrons, the rates of plasma clearance and incorporation of the label in triglycerides (TG), phospholipids (PL) and other lipids in the liver were compared at 5, 15 and 30 min. [14C]SA was cleared at a slightly faster rate (t1/2=7.0 min) than [14C]MA (t1/2=8.1 min) and [14C]LA (t1/2=8.0 min) (P<0.05). [14C]SA was accumulated in the liver at a significantly faster rate than [14C]MA and [14C]LA. At the peak (15 min) of hepatic uptake, 30.3% of [14C]SA, 26.2% of [14C]LA and 21.9% of [14C]MA were recovered in the liver. At 30 min, 33.5% of [14C]SA was taken up by the liver, whereas 27.8% of [14C]LA and only 15.2% of [14C]MA were removed. In the liver, the percentage of [14C]SA incorporated into PL steadily increased with time, whereas the percent‐age incorporated into TG decreased. [14C]SA was preferentially incorporated into PL at all time intervals, as compared with [14C]MA and [14C]LA. At 30 min, 38.6% of [14C]SA was found in PL, and only 5.2% of [14C]MA and 12.0% of [14C]LA were present in PL. A large proportion of hepatic [14C]MA remained unesterified (free fatty acid) throughout the 30‐min period, with a small proportion incorporated into PL and TG. Of the total liver14C radioactivity recovered at 30 min, 63.8% of [14C]MA, 48.8% of [14C]LA and 25.5% of [14C]SA were found unesterified. During 30 min, a significantly greater amount of [14C]MA (76.9%) was oxidized in both the liver and the peripheral tissue combined, compared with [14C]LA (64.7%) and [14C]SA (61.2%). A higher proportion of [14C]LA was incorporated into TG than into PL at all time intervals. No differences were noted in the relative distribution of14C in cholesterol and other lipids among the three fatty acids. Using labeled fatty acids incorporatedin vivo into chylomicrons, the present study demonstrated that SA, MA and LA are distinctly different in their metabolic behavior. During the initial 30 min after their entry into the blood, 92–95% of the fatty acids were cleared. During this early phase of metabolism, [14C]SA was preferentially utilized for liver PL synthesis, whereas [14C]LA was better incorporated into TG. [14C]MA was poorly incorporated into hepatic lipids, but was preferentially oxidized in the liver or utilized by the peripheral tissue.
Bile acid and very low density lipoprotein production by cultured hepatocytes from hypo‐ orhyperresponsive rabbits fedcholesterol
Authors: Podrez Evgeniy A. , Kosykh Vladimir A. , Lakeev Yuri V. , Kosenkov Evgeniy I. , Mambetisaeva Elvira T. , Repin Vadim S. , Sminov Vladimir N. , Miettinen Tatu A.
Published: November 2006

Two groups of rabbits, either hyperresponsive or hyporesponsive to dietary cholesterol, wereselected after ten weeks of cholesterol feeding (0.2 g cholesterol/kg body weight per day). Bile acids and very low density lipoprotein (VLDL) production were determined in primary hepatocyte cultures from control, hyper‐ and hyporesponsive rabbits. Free cholesterol and cholesteryl ester contents in hepatocytes of the hyperresponsive rabbits was significantly increased. In contrast, lipid composition in hepatocytes of the hyporesponders was similar to that of control cells. Cholic acid was the predominant bile acid in the culture medium of hepatocytes together with small amounts of chenodeoxycholic and deoxycholic acids. The rate of cholic acid production by hepatocytes in the hyporesponsive group was two times higher than that in the hyperresponsive group. Bile acid production by control hepatocytes was slightly higher than in the hyperresponsive group. In contrast, secretion of VLDL cholesteryl ester was significantly increased by hepatocytes of the hyperresponsive rabbits. Similar differences, in bile acid production were found between hypo‐ and hyperresponsive rabbits selected after five days of cholesterol feeding and subsequent maintenance on a low cholesterol diet for a period of one month. The results suggest that the increased rate of bile acid production could contribute to the apparent resistance of hyporesponders to the atherogenic diet.
Mouse alveolar surfactant: Characterization of subtypes prepared by differential centrifugation
Authors: Oulton M. , MacDonald J. , Janigan D. T. , Faulkner G. T.
Published: November 2006

To characterize the properties of alveolar surfactant susbfractions obtained from mouse lung by differential centrifugation, lavage fluid, following a preliminary centrifugation at 140 × g for 5 min to yield a cellular pellet (Pc), was sequentially cetrifuged at 10,000 ×g for 30 min, 60,000 ×g for 60 min and 100,000 ×g for 15 h; and the resultant pellets, respectively referred to as P10, P60 and P100, were harvested for electron microscopy, phospholipid analysis and surface tension measurements. Ultrastructural differences were observed, in that P10 contained large multilamellated structures which were typical of newly secreted surfactant, P100 contained small unilamellar vesicular structures, typical of catabolic end products of alveolar surfactant and P60 appeared to contain a mixture of structures present in P10 and P100 in addition to numerous, large unilamellar vesicles which were not present in either P10 or P100. Slight but significant differences were found in the phospholipid compositions of the three subfractions but not in the fatty acid composition of their phosphatidylcholine (PC) component. There were no significant differences in their disaturated PC/total PC ratios, but significant differences in their phospholipid/protein ratios. P60 had the highest proportion of phospholipid to protein. P10 and P60 demonstrated surface activity but P100 did not. Total alveolar surfactant phospholipid was evenly distributed among the three fractions. This pattern of distribution was significantly different from that observed in rabbit subfractions prepared by the same procedure. These data indicate that mouse alveolar surfactant consists of three distinct subfractions or subtypes which can be separately and quantitatively isolated by differential centrifugation. They also suggest that there may be species differences in the relative proportions of the individual subtypes present in normal adult lung.
The role of arginines in stabilizing the active open‐lid conformation ofRhizomucor miehei lipase
Authors: Holmquist Mats , Norin Martin , Hult Karl
Published: November 2006

Molecular dynamics simulations for the lid covering the active site ofRhizomucor miehei lipase [EC 3.1.1.3] postulated that, among other interactions, Arg86 in the lid stabilized the open‐lid conformation of the protein by multiple hydrogen bonding to the protein surface. Chemical modification of arginine residues inR. miehei lipase with 1,2‐cyclohexanedione or phenylglyoxal resulted in residual activities in the hydrolysis of tributyrin of 66 and 46%, respectively. Tryptic maps of native and phenylglyoxal‐reactedR. miehei lipase showed that Arg86 was the residue modified most, when the lipase was inhibited to the greatest extent. Guanidine, a structural analog to an arginine side chain, inhibited both the natibe enzyme and the arginine‐modified enzymes, resulting in residual activities of 26% as compared to the native enzyme. The inhibition was not an effect of enzyme denaturation. The native enzyme was also inhibited by 1‐ethylguanidine, benzamidine and urea, but to a lesser degree than by guanidine. Lipases fromHumicola lanuginosa and porcine pancreas in 100 mM guanidine showed residual activities of 88 and 70%, respectively. The lipases fromCandida antarctica, C. rugosa, Pseudomonas cepacia andP. fluoresences were not inhibited by guanidine. The inhibition ofR. miehei lipase by structural analogs of the arginine side chain and after chemical modification of arginine residues suggest a role of an arginine residue in stabilizing the active open‐lid conformation of the enzyme.
Systematic analysis of glycosphingolipids in the human gastrointestinal tract: Enrichment of sulfatides with hydroxylated longer‐chain fatty acids in the gastric and duodenal mucosa
Authors: Natomi Hitomi , Saitoh Toshihito , Sugano Kentaro , Iwamori Masao , Fukayama Masahisa , Nagai Yoshitaka
Published: November 2006

The composition of the glycosphingolipids of the human gastrointestinal tract was studied. The major neutral glycosphingolipids were ceramide monohexosides (e.g., GalCer, GlcCer), LacCer, Gb3Cer, Gb4Cer and more polar ones with more than four sugars, whereas neither Gg3Cer nor Gg4Cer were present. The acidic glycosphingolipids consisted of sulfatides and gangliosides such as GM3, GM1, GD3 and GD1a. Also a large amount of sulfatides was found in the gastric mucosa and duodenum. The concentrations of sulfatides in the fundic mucosa, antral mucosa and duodenum amounted to 416.0, 933.8 and 682.9 nmol/g of dry weight, respectively, exceeding those in the gastric mucosa and kidney of other mammals. The major molecular species of the sulfatides were identified as I3SO3‐GalCer with hydroxylated longer‐chain fatty acids based on the analyses by gas‐liquid chromatography and negative ion fast‐atom bombardment mass spectrometry. In contrast, gangliosides in these regions showed a tendency to be lower than sulfatides, and the molar ratios of sulfatides to gangliosides were about 2.0, whereas those in other parts were less than 0.5. A high content of sulfatides in the gastric and duodenal mucosa, where mucosa is easily insulted by acid, pepsin and bile salts, may be closely related to their roles in mucosal protection.
Kinetic study of the prooxidant effect of tocopherol. Hydrogen abstraction from lipid hydroperoxides by tocopheroxyls in solution
Authors: Mukai Kazuo , Sawada Kouhei , Kohno Yasuhiro , Terao Junji
Published: November 2006

A kinetic study of the prooxidant effect of vitamin E (tocopherol, TocH) has been carried out. The rates of hydrogen abstraction (k−1) from methyl linoleate hydroperoxide (ML‐OOH) by α‐tocopheroxyl (α‐Toc.) (1) and eight types of alkyl substituted Toc. radicals, (2–9) in benzene solution have been determined spectrophotometrically. The results show that the rate constants decrease as the total electron‐donating capacity of the alkyl substituents on the aromatic ring of Toc. increases. The k−1 value (5.0×10−1M−1s−1) obtained for α‐Toc. (1) was found to be about seven orders of magnitude lower than the k1 value (3.2×106M−1s−1) for the reaction of α‐TocH with peroxyl radical, which is well known as the usual radical‐scavenging reaction of α‐TocH. The above reaction rates (k−1) obtained were compared with those (k3) of methyl linoleate with Toc. (1–9) in benzene solution. The rates (k−1) were found to be about six times larger than those (k3) of the corresponding Toc.. The results suggest that both reactions may relate, to the prooxidant effect of α‐TocH at high concentrations in foods and oils. The effect of the phytyl side chain on the reaction rate, of Toc. in micellar dispersions has also been studied. We have measured the rate constant, k−1, for the reaction of phosphatidylcholine hydroperoxide with a Toc. radical in benzene,tert‐butanol and in Triton X‐100 micellar dispersions, and compared the observed k−1 values with the corresponding values for ML‐OOH.