Biliary phospholipid secretion is not required for intestinal absorption and plasma status of linoleic acid in mice

Biliary phospholipids have been hypothesized to be important for essential fatty acid homeostasis. We tested this hypothesis by investigating the intestinal absorption and the status of linoleic acid in mdr2 Pgp-deficient mice which secrete phospholipid-free bile. In mice homozygous (?/?) for disruption of the mdr2 gene and wild-type (+/+) mice, dietary linoleic acid absorption was determined by 72 h balance techniques. After enteral administration, [¹³C]-linoleic acid absorption was determined by measuring [¹³C]-linoleic acid concentrations in feces and in plasma. The status of linoleic acid was determined in plasma and in liver by calculating the molar percentage of linoleic acid and the triene:tetraene ratio. Although plasma concentration of [¹³C]-linoleic acid at 2 h after enteral administration was significantly lower in (?/?) compared to (+/+) mice (P≤0.05), net intestinal absorption of dietary linoleic acid or of [¹³C]-linoleic acid was similar in (+/+) and (?/?) mice. Molar percentage of linoleic acid and the triene:tetraene ratio were not different in whole plasma or in liver of (?/?) compared to (+/+) mice. Present data indicate that biliary phospholipids are involved in the rate of appearance in plasma of enterally administered linoleic acid, but are not required for net intestinal absorption or plasma status of linoleic acid.     

Fatty acid uptake in diabetic rat adipocytes

The effect of diabetic status and insulin on adipocyte plasma membrane properties and fatty acid uptake was examined. Studies with inhibitors and isolated adipocyte ghost plasma membranes indicated 9Z, 11E, 13E, 15Z-octatetraenoic acid (cis-parinaric acid) uptake was protein mediated. Cis-parinaric acid uptake was inhibited by trypsin treatment or incubation with phloretin, and competed with stearic acid. The initial rate, but not maximal uptake, of cis-parinaric acid uptake was enhanced two-fold in adipocytes from diabetic rats. Concomitantly, the structure and lipid composition of adipocyte ghost membranes was dramatically altered. However, the increased initial rate of cis-parinaric acid uptake in the diabetic adipocytes was not explained by membrane alterations or by a two-fold decrease in cytosolic adipocyte fatty acid binding protein (ALBP), unless ALBP stimulated fatty acid efflux. Thus, diabetic status dramatically altered adipocyte fatty acid uptake, plasma membrane structu re, lipid composition, and cytosolic fatty acid binding protein. (Mol Cell Biochem 167: 51-60, 1997)     

Increased plasma glutamic acid in a genetic model of epilepsy

A significant increase in the plasma levels of glutamic acid and a significant decrease in aspartic acid and taurine in epileptic patients and their first degree relatives was reported more than a decade ago and an underlying genetic basis for these amino acid changes was suggested. The main objective of the present study was to determine the plasma levels of glutamic acid, aspartic acid and taurine in El mice which are an inbred epileptic mutant mouse strain. The results show a significant increase in plasma glutamic acid but no changes in aspartic acid or taurine in the epileptic mice as compared to controls. The data provide the first evidence of a significant increase in plasma glutamic acid in an animal model of hereditary epilepsy and substantiate the hypothesis that a genetic defect underlies the elevated plasma glutamic acid levels in association with epilepsy. The findings are also compatible with neurochemical and neurophysiological evidence implicating glutamic acid in the mechanism of seizures.     

Regulation of the plasma amino acid profile by leucine via the system L amino acid transporter

Plasma concentrations of amino acids reflect the intracellular amino acid pool in mammals. However, the regulatory mechanism requires clarification. In this study, we examined the effect of leucine administration on plasma amino acid profiles in mice with and without the treatment of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) or rapamycin as an inhibitor of system L or mammalian target of rapamycin complex 1, respectively. The elevation of plasma leucine concentration after leucine administration was associated with a significant decrease in the plasma concentrations of isoleucine, valine, methionine, phenylalanine, and tyrosine; BCH treatment almost completely blocked the leucine-induced decrease in plasma amino acid concentrations. Rapamycin treatment had much less effects on the actions of leucine than BCH treatment. These results suggest that leucine regulates the plasma concentrations of branched-chain amino acids, methionine, phenylalanine, and tyrosine, and that system L amino acid transporters are involved in the leucine action.     

癫痫患者血浆中游离氨基酸水平分析

对７１例癫痫患者血浆中１８种游离氢基酸与３６例正常人血浆中游离氨基酸的水平对照。分析表明,抑制性氨基酸（γ－氨基丁酸、甘氨酸）水平明显升高的癫痫患者,其它氨基酸水平绝大多数低于正常人;兴奋性氨基酸（谷氨酸、天门冬氨酸、丙氨酸、谷氨酰胺）水平明显升高的癫痫患者,其它氨基酸水平低于正常人。     

Determination of the halothane metabolites trifluoroacetic acid and bromide in plasma and urine by ion chromatography

Halothane (CF₃CHClBr), a widely used volatile anesthetic, undergoes extensive biotransformation in humans. Oxidative halothane metabolism yields the stable metabolites trifluoroacetic acid and bromide which can be detected in plasma and urine. To date, analytical methodologies have either required extensive sample preparation, or two separate analytical procedures to determine plasma and urine concentrations of these analytes. A rapid and sensitive method utilizing high-performance liquid chromatography-ion chromatography (HPLC-IC) with suppressed conductivity detection was developed for the simultaneous detection of both trifluoroacetic acid and bromide in plasma and urine. Sample preparation required only ultrafiltration. Standard curves were linear (r²≥0.99) from 10 to 250 μM trifluoroacetic acid and 2 to 5000 μM bromide in plasma and 10 to 250 μM trifluoroacetic acid and 2 to 50 μM bromide in urine. The assay was applied to quantification of trifluoroacetic acid and bromide in plasma and urine of a patient undergoing halothane anesthesia.     

High-performance liquid chromatographic method for determination of vanillin and vanillic acid in human plasma, red blood cells and urine

A simple high-performance liquid chromatographic method was developed for the determination of vanillin and its vanillic acid metabolite in human plasma, red blood cells and urine. The mobile phase consisted of aqueous acetic acid (1%, v/v)–acetonitrile (85:15, v/v), pH 2.9 and was used with an octadecylsilane analytical column and ultraviolet absorbance detection. The plasma method demonstrated linearity from 2 to 100 μg/ml and the urine method was linear from 2 to 40 μg/ml. The method had a detection limit of 1 μg/ml for vanillin and vanillic acid using 5 μl of prepared plasma, red blood cells or urine. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of vanillin in patients undergoing treatment for sickle cell anemia.     

Cholesterol depletion inhibits fatty acid uptake without affecting CD36 or caveolin-1 distribution in adipocytes

Caveolin-1 and CD36 are plasma membrane fatty acid binding proteins that participate in adipocyte fatty acid uptake and metabolism. Both are associated with cholesterol-enriched caveolae/lipid rafts in the plasma membrane that are important for long chain fatty acid uptake. Depletion of plasma membrane cholesterol reversibly inhibited oleate uptake by adipocytes without altering the amount or the cell surface distribution of either caveolin-1 or CD36. Cholesterol levels thus regulate fatty acid uptake by adipocytes via a pathway that does not involve altered cell surface localization of caveolin-1 or CD36.     

Simple and sensitive fluorimetric liquid chromatography method for the determination of valproic acid in plasma

A simple and sensitive liquid chromatographic method is described for the analysis of valproic acid in human plasma. The method is based on the derivatization of valproic acid extracted from acidified plasma with 2-(2-naphthoxy)ethyl 2-(piperidino)ethanesulfonate. The resulting derivative is highly responsive to a fluorimetric detector (excitation at 230 nm and emission at 350 nm), giving a low detection limit of 0.6 microM (S/N = 3, 10 microl injected). The relative standard deviations of the method for intra- and inter-day analyses (n = 5) are below 3.3 and 4.1%, respectively. Toluene was used for the extraction of valproic acid from plasma and the toluene extract obtained was subjected to subsequent derivatization without solvent replacement. The simple method was applied to the analysis of valproic acid in plasma of dosed patients using only small amount of sample (10-50 microl plasma).     

Effects of antinutritional factors on plasma lipoprotein levels in Japanese flounder Paralichthys olivaceus

This study examined the effects of four types of antinutritional factor (phytic acid, stachyose, soy saponins and soy isoflavones) on lipoprotein levels in plasma of Japanese flounder Paralichthys olivaceus. A basal diet was prepared with fish meal as primary protein source, the other diets were supplemented with 0·2, 0·4 or 0·8% phytic acid, 0·4, 0·8 or 1·5% stachyose, 0·1, 0·35 or 0·7% soy saponins and 0·10, 0·35 or 0·70% soy isoflavones, by dry mass, in place of white flour in the basal diet. Total cholesterol (TC) and triglyceride (TG) levels in plasma of P. olivaceus were not affected by phytic acid or stachyose. In general, addition of 0·2-0·8% phytic acid or 0·4-1·5% stachyose decreased plasma high-density lipoprotein cholesterol (HDL-C) levels, increased plasma low-density lipoprotein cholesterol (LDL-C) levels, thereby increasing the LDL-C:HDL-C ratio. By contrast, supplementation with 0·35-0·7% soy saponins generally depressed plasma TC levels and the LDL-C:HDL-C ratio. Supplementation with 0·35-0·7% soy isoflavones, however, increased plasma TC and TG levels. These results indicate that soy saponins may be partly responsible for the cholesterol-lowering effects of soybean meal.     

High performance liquid chromatography–tandem mass spectrometry for the determination of bile acid concentrations in human plasma

We report a sensitive and robust method to determine cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), and their taurine- and glycine-conjugate concentrations in human plasma using liquid chromatography–tandem mass spectrometry. Activated charcoal was utilized to prepare bile acid-free plasma, which served as the biological matrix for the preparation of standard and quality control samples. Plasma sample preparation involved solid-phase extraction. A total of 16 bile acids and 5 internal standards were separated on a reverse column by gradient elution and detected by tandem mass spectrometry in negative ion mode. The calibration curve was linear for all the bile acids over a range of 0.005–5 μmol/L. The extraction recoveries for all the analytes fell in the range of 88–101%. Intra-day and inter-day coefficients of variation were all below 10%. A stability test showed that all the bile acids were stable in plasma for at least 6 h at room temperature, at least three freeze–thaw cycles, in the −70 °C or −20 °C freezer for 2 months, and also in the reconstitution solution at 8 °C for 48 h. Comparison of the matrix effect of bile acid-free plasma with that of real plasma indicated that the charcoal purification procedure did not affect the properties of charcoal-purified plasma as calibration matrix. This method has been used to determine the bile acid concentrations in more than 300 plasma samples from healthy individuals. In conclusion, this method is suitable for the simultaneous quantification of individual bile acids in human plasma.     

Dietary omega-6 fatty acid lowering increases bioavailability of omega-3 polyunsaturated fatty acids in human plasma lipid pools

BackgroundDietary linoleic acid (LA, 18:2n-6) lowering in rats reduces n-6 polyunsaturated fatty acid (PUFA) plasma concentrations and increases n-3 PUFA (eicosapentaenoic (EPA) and docosahexaenoic acid (DHA)) concentrations.ObjectiveTo evaluate the extent to which 12 weeks of dietary n-6 PUFA lowering, with or without increased dietary n-3 PUFAs, alters unesterified and esterified plasma n-6 and n-3 PUFA concentrations in subjects with chronic headache.DesignSecondary analysis of a randomized trial. Subjects with chronic headache were randomized for 12 weeks to (1) average n-3, low n-6 (L6) diet; or (2) high n-3, low n-6 LA (H3–L6) diet. Esterified and unesterified plasma fatty acids were quantified at baseline (0 weeks) and after 12 weeks on a diet.ResultsCompared to baseline, the L6 diet reduced esterified plasma LA and increased esterified n-3 PUFA concentrations (nmol/ml), but did not significantly change plasma arachidonic acid (AA, 20:4n-6) concentration. In addition, unesterified EPA concentration was increased significantly among unesterified fatty acids. The H3–L6 diet decreased esterified LA and AA concentrations, and produced more marked increases in esterified and unesterified n-3 PUFA concentrations.ConclusionDietary n-6 PUFA lowering for 12 weeks significantly reduces LA and increases n-3 PUFA concentrations in plasma, without altering plasma AA concentration. A concurrent increase in dietary n-3 PUFAs for 12 weeks further increases n-3 PUFA plasma concentrations and reduces AA.     

Determination of fenofibric acid in human plasma using automated solid-phase extraction coupled to liquid chromatography

The pharmacokinetic studies of fenofibrate require a rapid, selective and robust method to allow the determination of fenofibric acid, its active metabolite, in different biological matrixes (such as plasma, serum or urine). A new fully automated method for the determination of fenofibric acid in plasma has been developed, which involves the solid-phase extraction (SPE) of the analyte from plasma on disposable extraction cartridges (DECs) and reversed-phase HPLC with UV detection. The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with octadecyl silica was first conditioned with methanol and pH 7.4 phosphate buffer. A 0.8-ml volume of diluted plasma sample containing the internal standard (sulindac) was then applied on the DEC. The washing step was performed with the same buffer (pH 7.4). Finally, the analytes were successively eluted with methanol (1.0 ml) and 0.04 M phosphoric acid (1.0 ml). After a mixing step, 100 μl of the resultant extract was directly introduced into the HPLC system. The liquid chromatographic (LC) separation of the analytes was achieved on a Nucleosil RP-8 stationary phase (5 μm). The mobile phase consisted of a mixture of methanol and 0.04 M phosphoric acid (60:40, v/v). The analyte was monitored photometrically at 288 nm. The method developed was validated. In these conditions, the absolute recovery of fenofibric acid was close to 100% and a linear calibration curve was obtained in the concentration range from 0.25 to 20 μg/ml. The mean RSD values for repeatability and intermediate precision were 1.7 and 3.9% for fenofibric acid. The method developed was successfully used to investigate the bioequivalence between a micronized fenofibrate capsule formulation and a fenofibrate Lidose™ formulation.     

In vivo conversion of 18- and 20-C essential fatty acids in rats using the multiple simultaneous stable isotope method

An important question for mammalian nutrition is the relative efficiency of C18 versus C20 essential fatty acids (EFAs) for supporting the tissue composition of n-3 and n-6 pathway end products. One specific question is whether C22 EFAs are made available to tissues more effectively by dietary alpha-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) or by dietary eicosapentaenoic acid (20:5n-3) and dihomo-gamma-linolenic acid (20:3n-6). To address this question in a direct manner, four stable isotope compounds were given simultaneously in a novel paradigm. A single oral dose of a mixture of 2H5-18:3n-3, 13C-U-20:5n-3, 13C-U-18:2n-6, and 2H5-20:3n-6 was administered to rats given a defined diet. There was a preferential in vivo conversion of arachidonic acid (20:4n-6) to docosatetraenoic acid (22:4n-6) and of 22:4n-6 to n-6 docosapentaenoic acid (22:5n-6) when the substrates originated from the C18 precursors. However, when the end products docosahexaenoic acid (22:6n-3) or 22:5n-6 were expressed as the total amount in the plasma compartment divided by the dosage, this parameter was 11-fold greater for 20:5n-3 than for 18:3n-3 and 14-fold greater for 20:3n-6 than for 18:2n-6. Thus, on a per dosage basis, the total amounts of n-3 and n-6 end products accreted in plasma were considerably greater for C20 EFA precursors relative to C18.     

硝酸银硅胶柱层析分离血浆不饱和脂肪酸

目的:利用硝酸银硅胶填料有效分离出血浆混合脂肪酸中的亚油酸。方法:通过改进的FOLCH法提取血浆总脂后,再采用皂化、酸化水解的方法将总脂转化为混合脂肪酸。用ghosh法将硅胶改性为硝酸银硅胶后,以亚油酸为对象,通过静态吸附试验了解硝酸银硅胶对不饱和脂肪酸的吸附特性,采用柱层析的方法分离血浆混合脂肪酸中的亚油酸。结果:血浆与有机溶剂在1∶5时既能有效萃取血浆总脂,用正己烷∶二氯甲烷∶乙醚=89∶10∶1作为洗脱剂,将洗脱液甲酯化后进行GC和GC-MS检测,硝酸银硅胶柱的洗脱液中亚油酸的纯度60.74%,硅胶柱的为23.65%,不饱和脂肪酸得到了较好的纯化。     

Determination of hexahydrophthalic acid and methylhexahydrophthalic acid in plasma after derivatisation with pentafluorobenzyl bromide using gas chromatography and mass spectrometric detection

A method for the simultaneous determination of hexahydrophthalic acid (HHP acid) and methylhexahydrophthalic acid (MHHP acid) in human plasma was developed. The procedure was a rapid, single step extractive derivatisation with pentafluorobenzyl bromide as the derivatisation agent. The formed pentafluorobenzyl esters were analysed by gas chromatography-mass spectrometry in negative ion chemical ionisation mode with ammonia as the moderating gas. Deuterium-labeled HHP acid and MHHP acid were used as internal standards. The detection limit was 0.4 ng/ml for HHP acid (m/z 153) and 0.3 ng/ml for MHHP acid (m/z 365). The within-day precision of the method was between 2 and 3% and the between-day precision was between 3 and 12%. The overall recovery was between 65 and 83%. A comparison between HHP acid determinations with a previous and this method showed that the methods gave similar results. The method was applicable for analysis of plasma from occupationally exposed workers.     

Acid Lability of Metabolites of 2-Deoxyglucose in Rat Brain: Implications for Estimates of Kinetic Parameters of Deoxyglucose Phosphorylation and Transport Between Blood and Brain

The steady-state brain/plasma distribution ratios of [14C]deoxyglucose ([14C]DG) for hypoglycemic rats previously determined by measurement of DG concentrations in neutralized acid extracts of freeze-blown brain and plasma exceeded those predicted by simulations of kinetics of the DG model. Overestimation of the true size of the precursor pool of [14C]DG for transport and phosphorylation could arise from sequestration of [14C]DG within brain compartments and/or instability of metabolites of [14C]DG and regeneration of free [14C]DG during the experimental period or extraction procedure. In the present study, the concentrations of [14C]DG and glucose were compared in samples of rat brain and plasma extracted in parallel with perchloric acid or 65% ethanol containing phosphate-buffered saline. The concentrations of both hexoses in acid extracts of brain were higher than those in ethanol, whereas hexose contents of plasma were not dependent on the extraction procedure. The magnitude of overestimation of DG content (about 1.2-to fourfold) varied with glucose level and was highest in extracts isolated from hypoglycemic rats; contamination of the [14C]DG fraction with 14C-labeled nonacidic metabolites also contributed to this overestimation. Glucose concentrations in acid extracts of brain exceeded those of the ethanol extracts by less than 40% for normal and hypoglycemic rats.     

Determination of membrane cholesterol partition coefficient using a lipid vesicle-cyclodextrin binary system: effect of phospholipid acyl chain unsaturation and headgroup composition

Lateral domain or raft formation in biological membranes is often discussed in terms of cholesterol-lipid interactions. Preferential interactions of cholesterol with lipids, varying in headgroup and acyl chain unsaturation, were studied by measuring the partition coefficient for cholesterol in unilamellar vesicles. A novel vesicle-cyclodextrin system was used, which precludes the possibility of cross-contamination between donor-acceptor vesicles or the need to modify one of the vesicle populations. Variation in phospholipid headgroup resulted in cholesterol partitioning in the order of sphingomyelin (SM) > phosphatidylserine > phosphatidylcholine (PC) > phosphatidylenthanolamine (PE), spanning a range of partition DeltaG of -1181 cal/mol to +683 cal/mol for SM and PE, respectively. Among the acyl chains examined, the order of cholesterol partitioning was 18:0(stearic acid),18:1n-9(oleic acid) PC > di18:1n-9PC > di18:1n-12(petroselenic acid) PC > di18:2n-6(linoleic acid) PC > 16:0(palmitic acid),22:6n-3(DHA) PC > di18:3n-3(alpha-linolenic acid) PC > di22:6n-3PC with a range in partition DeltaG of 913 cal/mol. Our results suggest that the large differences observed in cholesterol-lipid interactions contribute to the forces responsible for lateral domain formation in plasma membranes. These differences may also be responsible for the heterogeneous cholesterol distribution in cellular membranes, where cholesterol is highly enriched in plasma membranes and relatively depleted in intracellular membranes.     

Uric Acid: The Oxidant-Antioxidant Paradox

Uric acid, despite being a major antioxidant in the human plasma, both correlates and predicts development of obesity, hypertension, and cardiovascular disease, conditions associated with oxidative stress. While one explanation for this paradox could be that a rise in uric acid represents an attempted protective response by the host, we review the evidence that uric acid may function either as an antioxidant (primarily in plasma) or pro-oxidant (primarily within the cell). We suggest that it is the pro-oxidative effects of uric acid that occur in cardiovascular disease and may have a contributory role in the pathogenesis of these conditions.     

肺癌患者胸水游离氨基酸分析

本文对11例肺癌患者胸水13种游离氨基酸作了分析,并与28例正常人血浆游离氨基酸水平作了对照,结果表明:肺癌患者胸水的必需及非必需氨基酸普遍高于正常人血浆游离氨基酸,但其胸水谷氨酰胺水平则明显低于正常人血浆水平。