Folch Method Research Articles

Accelerated Fatty Acid Oxidation in Muscle Averts Fasting-induced Hepatic Steatosis in SJL/J Mice

The accumulation of triglycerides (TG) in the liver, designated hepatic steatosis, is characteristically associated with obesity and insulin resistance, but it can also develop after fasting. Here, we show that fasting-induced hepatic steatosis is under genetic control in inbred mice. After a 24-h fast, C57BL/6J mice and SJL/J mice both lost more than 20% of body weight and approximately 60% of total body TG. In C57BL/6J mice, TG accumulated in liver, producing frank steatosis. In striking contrast, SJL/J mice failed to accumulate any hepatic TG even though they lost nearly as much adipose tissue mass as the C57BL/6J mice. Mice from five other inbred strains developed fasting-induced steatosis like the C57BL/6J mice. Measurements of the uptake of free fatty acids (FA) in vivo and in vitro demonstrated that SJL/J mice were protected from steatosis because their heart and skeletal muscle took up and oxidized twice as much FA as compared with C57BL/6J mice. As a result of this muscle diversion, serum-free FA and ketone bodies rose much less after fasting in SJL/J mice as compared with C57BL/6J mice. When livers of SJL/J and C57BL/6J mice were perfused with similar concentrations of FA, the livers took up and esterified similar amounts. We conclude that SJL/J mice express one or more variant genes that lead to enhanced FA uptake and oxidation in muscle, thereby sparing the liver from FA overload in the fasting state.

Trans 지방과 쌀, 콩을 첨가한 빵의 섭취가 건강한 성인 남녀의 혈장 지질 및 지방산 조성에 미치는 영향

It has been reported that trans fat (tFA) may have adverse or beneficial effect depending upon the position and number of double bonds. The presence of tFA in human tissues and fluids is related to dietary intake, intestinal absorption, metabolism and storage, exchanges among compartments. This study investigated the effect of breads containing tFA, soybean or rice on postprandial plasma fatty acid and lipid composition. 33 healthy volunteers were divided into 3 groups and fed soybean bread, rice bread or wheat bread groups containing equivalent amounts of tFA (elaidic acid rich, 3.75 g/day), respectively. Postprandial lipid profiles at 0, 1, 2, 3 and 4 hours after a respective meal were studied. Plasma fatty acid was extracted by the method of Folch and methyl ester of fatty and prepared by acid transmethylation and analyzed by Gas Chromatography. Peaks were identified using pure reference compounds and quantified. Postprandial data indicated that consumption of soybean and rice breads with 3.75 g tFA retarded the appearance of C18:1 and C18:2 tFA in plasma lipid compared to that of wheat bread. Futhermore, soybean and rice bread groups showed lower plasma saturated fatty acid levels than wheat bread group. Postprandial TG level was significantly lowered in soybean bread group compared to that of rice and wheat bread groups. These results imply that soybean bread with high dietary fiber content and biologically active substances may inhibit or delay lipid absorption.

Conjugated Linoleic Acid in Rumen Liquor: Their Relation with Other Carbon 18 Fatty Acids and Rumen pH

This study was focused on the quantitative analysis of Conjugated Linoleic Acids (CLAs) in rumen liquor and assessment of the relationship between CLAs and other carbon 18 fatty acids and rumen pH of cattle slaughtered for meat consumption. The fatty acids were extracted using modified Folch method and determined by gas chromatography. The mean (n = 22) level of CLAs in the rumen liquor was 14.97 ±7.46 mg/100 mL. Cis-9, trans-11(c9, t11) was the dominant isomer of CLA followed by trans-10,cis-12 (t10, c12). The level of CLAs was positively correlated with the concentration of octadecatrienoic (18:3), cis-9, cis-12 octadecadienoic (18:2), trans-11octadecenoic (18:1) and octadecenoic (18:0) acids. Ruminal pH was positively correlated with the amount of c9, t11 and total CLA and negatively correlated with t10, c12 CLA isomer. All these correlations and relationships indicated that CLAs production in the rumen is dependent on availability of carbon 18 fatty acids and pH of the rumen liquor.

Optimization of an Oil Extraction Process for Algae from the Treatment of Manure Effluent

AbstractIncreasing interest in the coupling of biological wastewater treatment processes with the generation of value‐added products (such as oil containing ω‐3 fatty acids (FA)) has stimulated efforts in adapting extraction methods for treatment byproducts. This study’s objective was to compare a high temperature/pressure extraction method (accelerated solvent extraction) (ASE) and a manual extraction method (modified Folch extraction) with regard to their ability to extract total oil from three algae samples from the treatment of dairy manure effluent. The efficiency of total oil and FA extraction with three solvents (chloroform/methanol, isopropanol/hexane, and hexane) was also evaluated using the ASE method. Results showed that the ASE method yielded higher values for total oil content compared to the Folch method but similar values for FA content and composition after four extraction cycles with chloroform/methanol. However, the ASE method yielded much higher amounts of FA in the first cycle (85–95% of total extracted) compared to the Folch method (44–55% of total extracted in the first cycle). As expected, the extraction efficiency of the ASE method for FA was dependent on the extraction solvent. FA content values using ASE with chloroform/methanol > isopropanol/hexane > hexane. FA content values using the Folch method or ASE with chloroform/methanol were not significantly influenced by sample particle size within the size range of 0.1–1 mm.

The Kap60-Kap95 Karyopherin Complex Directly Regulates Phosphatidylcholine Synthesis

Phosphatidylcholine is the major phospholipid in eukaryotic cells. There are two main pathways for the synthesis of phosphatidylcholine: the CDP-choline pathway present in all eukaryotes and the phosphatidylethanolamine methylation pathway present in mammalian hepatocytes and some single celled eukaryotes, including the yeast Saccharomyces cerevisiae. In S. cerevisiae, the rate-determining step in the synthesis of phosphatidylcholine via the CDP-choline pathway is catalyzed by Pct1. Pct1 converts phosphocholine and CTP to CDP-choline and pyrophosphate. In this study, we determined that Pct1 is in the nucleoplasm and at endoplasmic reticulum and nuclear membranes. Pct1 directly interacts with the alpha-importin Kap60 via a bipartite basic region in Pct1, and this region of Pct1 was required for its entry into the nucleus. Pct1 also interacted with the beta-importin Kap95 in cell extracts, implying a model whereby Pct1 interacts with Kap60 and Kap95 with this tripartite complex transiting the nuclear pore. Exclusion of Pct1 from the nucleus by elimination of its nuclear localization signal or by decreasing Kap60 function did not affect the level of phosphatidylcholine synthesis. Diminution of Kap95 function resulted in almost complete ablation of phosphatidylcholine synthesis under conditions where Pct1 was extranuclear. The beta-importin Kap95 is a direct regulator membrane synthesis.

TNF-α plays a role in hepatocyte apoptosis in Niemann-Pick type C liver disease

Niemann-Pick type C (NPC) is a fatal autosomal recessive lysosomal storage disease clinically characterized by neurodegeneration and liver disease. Heterogeneous mutations in the NPC1 and NPC2 genes cause impaired egress of free cholesterol from lysosomes, leading to accumulation of cholesterol and glycosphingolipids. Key features of NPC liver disease include hepatic apoptosis, inflammation, and fibrosis. It is unclear what signaling events regulate these disease processes in NPC. We hypothesize that tumor necrosis factor alpha (TNF-alpha), which is involved in both proinflammatory and apoptotic signaling cascades, is a key mediator of inflammation, apoptosis, and fibrosis in NPC liver disease. In this study, we evaluated the role of TNF-alpha signaling in NPC liver disease by utilizing NPC1-specific antisense oligonucleotides to knock down NPC1 expression in control and TNF-alpha knockout mice. In the absence of TNF-alpha, NPC1 knockdown produced liver disease with significantly less inflammation, apoptosis, and fibrosis.

Disorders of phospholipids metabolism in the brain of white rats during experimental liver cirrhosis and corrective effect of antioxidant factors

1 The goal of the present work was to study the peculiarities of the phospholipids‐phospholipid (PL-PL) ratio in white rat brain in CCl 4 -induced liver cirrhosis (LC) and the corrective effect of antioxidant factors such as α -tocopherol ( α -TPh) and sodium thiosulfate (STS). The experiments were performed with outbred white male rats weighing 180‐200 g. Experimental LC was induced by i.p. injection of 0.3 ml/kg CCl 4 , which was performed twice a week for 20 days. The PLs were extracted by Folch method using the Karagezyan modification. The PL quantity was measured by the Lowry method in µ g of mineralized lipid phosphorus per mg of protein. The analysis of the results obtained indicates significant quantitative and qualitative changes in the phospholipid contents in the samples studied. The quantity of lysophosphatidylcholine (LPC) in the brain of the diseased animals increased by 47%, which could be the result of intensification of phosphatidylcholine (PC) deacylation. A decrease in the PC contents (11%) was accompanied by a simultaneous decrease in phosphatidylserine (PS) and phosphatidylethanolamine (PE) levels by 14% and 12%, respectively. A decrease in the PC, PS, and PE contents may related to the fact of their rapid oxidation by free radicals in the strong processes of lipid peroxidation. The CCl 4 -induced LC is also accompanied by an increase in the content of PLs that have slow metabolism in brain, such as sphingomyelins (SPMs), the main lipid components of myelin fraction. The SPMs are very saturated lipids, which do not possess polyenic acids. Hence, a high quantity of these lipids results in the expressed inflexibility of membranes, i.e., in increased microviscosity of the lipid phase of the membrane, which is characterized by suppressed Na, KATPase activity. Cardiolipin CL, together with PS and monophosphoinositide (MPI), provides a sufficient level of hydrophobicity, lipid-dependent activity of mitochondrial enzymes of the electron transport chain in the indicated fractions of cells, and the respiratory function of cells as a whole. The content of cardiolipin (CL) in the brain is not great; however, an increase of the content of this lipid is important. It is known that the CLs are the carriers of a negative charge, hence, they are capable of fulfilling an ionophoric function, including an effect on

Specific features of sacricin action on the disturbances in phospholipid metabolism during seizures

1 is a brain disease characterized by strong hyperactivity of neuronal populations. The goal of our research was to study different aspects of lipid metabolism in different phases of convulsive body response, namely, before and after a sacricin injection. Sacricin, a derivative of polycyclic carboxylic acid, possesses rather large anticonvulsant effect. The phospholipid (PL) extraction was performed with the use of Folch method [1], using Karagezyan’s modification [2]. PLs were fractionated by one-dimensional ascending chromatography in a thin layer of silica gel (Merk, Germany) using the solvent system chloroform : methanol : ammonia (65 : 35 : 5). Convulsive activity is characterized by disturbances of membrane-bound enzyme functional activity, a decrease in the number of receptors, increase in “flip-flop” transfers of phospholipids, and probable hydrolysis of phosphatidylinositols, which are involved in nerve impulse transfer and affect transport of PLs and diglyceride, which are secondary messengers [3, 4] responsible for maintenance of the functional activity of a cell. Taking into consideration the role of lipids in the structure‐functional organization of biological membranes [5, 6], we studied PL‐PL conversions in the brain cortex and cerebellum of rats during pentylenetetrazol seizures in the presence and absence of sacricin. Based on the presented results, we assumed the following: the convulsions enhanced the activity of phospholipase A 2 , which catalyzes reactions of PL deacyla1 Corresponding author; address: Hasratyan St. 7, Yerevan 0014, Armenia; e-mail: art79@rambler.ru tion and the formation of nonesterified fatty acids, which are substrates for the formation of a large quantity of lipid peroxides. We observed qualitative and quantitative shifts in the PL-PL ratio. Especially strong shifts were observed in the contents of phosphatidylcholines, phosphatidylserines, and mainly in lysophosphatidylcholines, which are the products of lysis of phosphatidylcholines. Injection of sacrisin strongly normalized PL metabolism and lipid peroxidation. The molecular mechanisms of the normalizing effect of this drug are associated with systems that regulate the reactions of PL‐PL interactions and peroxidation of lipids.

Effects of a black rice extract ( Oryza sativa L. indica) on cholesterol levels and plasma lipid parameters in Wistar Kyoto rats

This study was designed to evaluate the efficacy of an anthocyanin pigmented rice (e.g. black rice) to mitigate the onset of hypercholesterolemia in rats-fed atherogenic diets. Male Wistar ( n = 10/group) rats were fed with atherogenic diets containing 0.5% cholesterol in the presence and in the absence of bile salt (e.g. 0.05% cholic acid) along with a standardized black rice extract (BRE) (e.g. 3%, w/w). All animals were individually housed in stainless steel cages and fed with the experimental diets during a 12-h period for 10 weeks. Body weights of rats were measured every week of the experiment. After 10 weeks fed on experimental diets, rats were sacrificed and plasma total cholesterol, HDL and LDL cholesterol and triacylglycerols were measured immediately. The total cholesterol (TC) content in the liver, heart and aorta, and the concentration of triacylglycerol (TAG) were measured after lipid extraction using Folch method. Rats fed with 0.5% cholesterol containing diets which also included bile salt exhibited a considerably more severe hypercholesterolemia than counterparts fed diets containing only 0.5% cholesterol. The inclusion of the BRE in diets significantly ( p < 0.05) decreased the level of TC, LDL–TC and TAG in plasma of rats-fed control diets that either contained or were absent in bile salt ( p < 0.05). There were no differences in HDL-level. Liver crude lipids and total cholesterol levels were also significantly ( p < 0.05) decreased in experimental groups relative to the control group in both experiments. Thus, supplementation of atherogenic experimental diets with BRE effectively decreased lipid levels in hypercholesterolemic rats. In lieu of the mixture of bioactive components present in BRE, it is possible that more than one mechanism underlying this reduction in lipids is involved.

NADPH Oxidase-dependent Generation of Lysophosphatidylserine Enhances Clearance of Activated and Dying Neutrophils via G2A

Exofacial phosphatidylserine (PS) is an important ligand mediating apoptotic cell clearance by phagocytes. Oxidation of PS fatty acyl groups (oxPS) during apoptosis reportedly mediates recognition through scavenger receptors. Given the oxidative capacity of the neutrophil NADPH oxidase, we sought to identify oxPS signaling species in stimulated neutrophils. Using mass spectrometry analysis, only trace amounts of previously characterized oxPS species were found. Conversely, 18:1 and 18:0 lysophosphatidylserine (lyso-PS), known bioactive signaling phospholipids, were identified as abundant modified PS species following activation of the neutrophil oxidase. NADPH oxidase inhibitors blocked the production of lyso-PS in vitro, and accordingly, its generation in vivo by activated, murine neutrophils during zymosan-induced peritonitis was absent in mice lacking a functional NADPH oxidase (gp91phox-/-). Treatment of macrophages with lyso-PS enhanced the uptake of apoptotic cells in vitro, an effect that was dependent on signaling via the macrophage G2A receptor. Similarly, endogenously produced lyso-PS also enhanced the G2A-mediated uptake of activated PS-exposing (but non-apoptotic) neutrophils, raising the possibility of non-apoptotic mechanisms for removal of inflammatory cells during resolution. Finally, antibody blockade of G2A signaling in vivo prolonged zymosan-induced neutrophilia in wild-type mice, whereas having no effect in gp91phox-/- mice where lyso-PS are not generated. Taken together, we show that lyso-PS are modified PS species generated following activation of the NADPH oxidase and lyso-PS signaling through the macrophage G2A functions to enhance existing receptor/ligand systems for optimal resolution of neutrophilic inflammation.

Glyceroneogenesis Is the Dominant Pathway for Triglyceride Glycerol Synthesis in Vivo in the Rat

Triglyceride synthesis in mammalian tissues requires glycerol 3-phosphate as the source of triglyceride glycerol. In this study the relative contribution of glyceroneogenesis and glycolysis to triglyceride glycerol synthesis was quantified in vivo in adipose tissue, skeletal muscle, and liver of the rat in response to a chow diet (controls), 48-h fast, and lipogenic (high sucrose) diet. The rate of glyceroneogenesis was quantified using the tritium ([(3)H(2)]O) labeling of body water, and the contribution of glucose, via glycolysis, was determined using a [U-(14)C]glucose tracer. In epididymal and mesenteric adipose tissue of control rats, glyceroneogenesis accounted for approximately 90% of triglyceride glycerol synthesis. Fasting for 48 h did not alter glyceroneogenesis in adipose tissue, whereas the contribution of glucose was negligible. In response to sucrose feeding, the synthesis of triglyceride glycerol via both glyceroneogenesis and glycolysis nearly doubled (versus controls); however, glyceroneogenesis remained quantitatively higher as compared with the contribution of glucose. Enhancement of triglyceride-fatty acid cycling by epinephrine infusion resulted in a higher rate of glyceroneogenesis in adipose tissue, as compared with controls, whereas the contribution of glucose via glycolysis was not measurable. Glyceroneogenesis provided the majority of triglyceride glycerol in the gastrocnemius and soleus. In the liver the fractional contribution of glyceroneogenesis remained constant (approximately 60%) under all conditions and was higher than that of glucose. Thus, glyceroneogenesis, in contrast to glucose, via glycolysis, is quantitatively the predominant source of triglyceride glycerol in adipose tissue, skeletal muscle, and liver of the rat during fasting and high sucrose feeding.

Static–dynamic sequential superheated liquid extraction of phenols and fatty acids from alperujo

Superheated liquids of different polarity have been used for sequential extraction of fatty acids and phenols from alperujo. Multivariate methodology has been used to optimise the static-dynamic extraction. Forty-two minutes are required to complete extraction (20 mg/kg of fatty acids and up to 2,200 mg/kg of hydroxytyrosol in the raw material used). The efficacy of the extraction has been demonstrated and compared with that of conventional methods (Folch and stirring-based methods for fatty acids and phenols, respectively), which needed 4.5 and 24 h for the extraction of fatty acids and phenols, respectively. The non-polar and polar extracts were injected into GC-MS and HPLC-MS-MS equipment, respectively, for individual separation-quantification of the target compounds. The simplicity of the experimental setup and the low costs of the raw material make the proposed method advisable when extraction of both fractions is required.

Ganglioside composition of differentiated Caco-2 cells resembles human colostrum and neonatal rat intestine

Gangliosides are glycosphingolipids found in cell membranes and human milk with important roles in cell proliferation, differentiation, growth, adhesion, migration, signalling and apoptosis. Similar changes in ganglioside composition occur during embryonic development, lactation and cancer cell differentiation. It is not known, however, whether ganglioside compositional changes that occur in differentiating colon cancer cells reflect changes that occur during intestinal development. The Caco-2 cell line is commonly used to study physiological and pathophysiological processes in the small intestine and colon. Therefore, to examine this question, undifferentiated and differentiated Caco-2 cells were grown and total lipid was extracted from cell supernatant fractions using the Folch method. The upper aqueous phase containing gangliosides was collected and purified. Total gangliosides were measured as ganglioside-bound N-acetyl neuraminic acid, while individual ganglioside content was quantified via a colorimetric assay for sialic acid and scanning densitometry. The total ganglioside content of differentiated Caco-2 cells was 2.5 times higher compared with undifferentiated cells. Differentiated Caco-2 cells had significantly more (N-acetylneuraminyl) 2-galactosylglucosyl ceramide (GD3) and polar gangliosides, and a lower N-acetylneuraminylgalactosylglucosylceramide (GM3):GD3 ratio than undifferentiated cells. The present study demonstrates that the total ganglioside content and individual ganglioside composition of differentiated Caco-2 cells are similar to those of human colostrum and neonatal rat intestine. Differentiated Caco-2 cells may therefore be an alternative model for studying physiological and pathological processes in the small intestine and colon, and may help to elucidate possible functions for specific gangliosides in development and differentiation. Further research using more sensitive techniques of ganglioside analysis is needed to confirm these findings.

Activation of Liver X Receptors Prevents Statin-induced Death of 3T3-L1 Preadipocytes

The biological functions of liver X receptors (LXRs) alpha and beta have primarily been linked to pathways involved in fatty acid and cholesterol homeostasis. Here we report a novel role of LXR activation in protecting cells from statin-induced death. When 3T3-L1 preadipocytes were induced to differentiate by standard isobutylmethylxanthine/dexamethasone/insulin treatment in the presence of statins, they failed to differentiate and underwent massive apoptosis. The simultaneous addition of selective LXR agonists prevented the statin-induced apoptosis. By using mouse embryo fibroblasts from wild-type (LXRalpha+/+/LXRbeta+/+), LXRalpha knock-out mice (LXRalpha(-/-)/LXRbeta+/+), LXRbeta knock-out mice (LXRalpha+/-/LXRbeta(-/-)), and LXR double knock-out mice (LXRalpha(-/-)/LXRbeta(-/-)) as well as 3T3-L1 cells transduced with retroviruses expressing either wild-type LXRalpha or a dominant negative version of LXRalpha, we demonstrate that the response to LXR agonists is LXR-dependent. Interestingly, LXR-mediated rescue of statin-induced apoptosis was not related to up-regulation of genes previously shown to be involved in the antiapoptotic action of LXR. Furthermore, forced expression of Bcl-2 did not prevent statin-induced apoptosis; nor did LXR action depend on protein kinase B, whose activation by insulin was impaired in statin-treated cells. Rather, LXR-dependent rescue of statin-induced apoptosis in 3T3-L1 preadipocytes required NF-kappaB activity, since expression of a dominant negative version of IkappaBalpha prevented LXR agonist-dependent rescue of statin-induced apoptosis. Thus, the results presented in this paper provide novel insight into the action of statins on and LXR-dependent inhibition of apoptosis.

Comparison of extraction and derivatization methods for fatty acid analysis in solid environmental matrixes

This study involved comparison of different extraction and derivatization methods for determining FAs in soil and in four highly organic matrixes (cattle manure, pig slurry, compost, and vermicompost), by application of a multifactor categorical design. Although some studies have been carried out regarding the application of FA analysis to highly organic matrixes, comparison and verification are still required to test which methods of extraction and derivatization of FAs function best for these matrixes. We compared three extraction methods (one in which the same extraction mixture as used in the Folch method was employed, a modification of the Bligh and Dyer method, and a microwave-assisted extraction) and two derivatization procedures (alkaline methanolysis and derivatization with trimethylsulfonium hydroxide (TMSH)). The highest yields of FAs belonging to different structural classes, and of individual FAs used as microbial biomarkers were obtained by application of the same extraction mixture as in the Folch method and use of TMSH as derivatization agent. These methods also involved a significant reduction in the complexity and time involved in sample preparation.

Chemical analysis of atherosclerotic plaque cholesterol combined with histology of the same tissue

Sensitive method for chemical analysis of free cholesterol (FC) and cholesterol esters (CE) was developed. Mouse arteries were dissected and placed in chloroform-methanol without tissue grinding. Extracts underwent hydrolysis of cholesteryl esters and derivatization of cholesterol followed by liquid chromatography/mass spectrometry (LC/MS/MS) analysis. We demonstrated that FC and CE could be quantitatively extracted without tissue grinding and that lipid extraction simultaneously worked for tissue fixation. Delipidated tissues can be embedded in paraffin, sectioned, and stained. Microscopic images obtained from delipidated arteries have not revealed any structural alterations. Delipidation was associated with excellent antigen preservation compatible with traditional immunohistochemical procedures. In ApoE(-/-) mice, LC/MS/MS revealed early antiatherosclerotic effects of dual PPARalpha,gamma agonist LY465606 in brachiocephalic arteries of mice treated for 4 weeks and in ligated carotid arteries of animals treated for 2 weeks. Reduction in CE and FC accumulation in atherosclerotic lesions was associated with the reduction of lesion size. Thus, a combination of LC/MS/MS measurements of CE and FC followed by histology and immunohistochemistry of the same tissue provides novel methodology for sensitive and comprehensive analysis of experimental atherosclerotic lesions.

Cholesterol oxidation in traditional Mexican dried and deep-fried food products

A number of animal and in vitro studies have demonstrated that cholesterol oxidation products (COPs) are the primary factor that triggers the atherosclerotic lesion. Furthermore, it has been recognized that several COPs have carcinogenic, cytotoxic and mutagenic effects. Although some works have reported the presence of COPs in a number of dried and deep-fried foods, other high-risk food products have not yet been evaluated. The aim of this study was to analyze the content of COPs of three widely consumed, typical Mexican food products: deep-fried pork rinds ( chicharrón), dried beef ( machaca) and sun-dried shrimp, which are subjected to severe thermal treatments and have relatively high cholesterol content. Lipids were extracted according to Folch's method, then subjected to cold saponification, purified by aminopropyl solid-phase extraction, silanized and analyzed by gas chromatography. The most abundant COPs found in all samples were 7 α-hydroxycholesterol, 7 β-hydroxycholesterol and 7-ketocholesterol. The total amount of COPs varied from 45 to 57 mg/kg in chicharrón and machaca, whereas the sun-dried shrimp showed higher COPs content (131–254 mg/kg). The extent of cholesterol oxidation of chicharrón, machaca and sun-dried shrimp were 2.3%, 5.1% and 14.5%, respectively.

Effect of absorbent in solid‐phase extraction on quantification of phospholipids in palm‐pressed fiber

AbstractPalm‐pressed fiber (PPF) is a by‐product of palm oil milling and it has been found to contain a high percentage of phospholipids (PL). The aim of this work was to evaluate the best solid‐phase extraction (SPE) method to purify PL from PPF. The purified PL were analyzed using high‐performance liquid chromatography (HPLC) with an evaporative light‐scattering detector (ELSD). The oil was extracted from PPF using the Folch method and classes of PL were purified using SPE. Different solvent phases and normal‐phase SPE cartridges [silica (Si), aminopropyl‐bonded silica (NH2) and diol‐bonded silica (2OH)] at the same ratio of oil to sorbent mass were used to study the separation of PL from the extracted oil. The recovery of each standard PL was performed in a model oil system with the same amount of standard PL being added, and the repeatability of the SPE method was investigated. The isolation of PL by SPE diol cartridge and subsequent analysis by HPLC/ELSD have shown to be an accurate and reproducible analytical method for the determination of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and lysophosphatidylcholine in PPF. This method provided a high yield and rapid separation of PL in PPF with less contamination from other lipid groups.

Diallyl Sulfide Inhibits Lipid Peroxidation by increasing Superoxide dismutase and Glutathione S Transferase Gene Expression in the Liver of Male Sprague Dawley Rats.

Diethylstilbestrol (DES), a synthetic estrogen, is associated with liver cancer in rats and humans. DES undergoes redox cycling producing free radicals which can react with lipids causing lipid peroxidation. Lipid peroxides causes DNA damage, mutations and ultimately cancer. Dially sulfide has been shown to inhibit various types of cancers presumably by metabolic modulation. We hypothesize that DAS will inhibit lipid peroxidation by increasing gene expression of superoxide dismutase (SOD) and Glutathione S Transferase (GST) in the liver of male Sprague Dawley rats. To test this hypothesis four groups of five male Sprague Dawley rats were treated as follows: (1) control (corn oil), (2) 50 mg/kg DAS (3) 50 mg/kg DES, (4) 50 mg/kg DAS, 50mg/kg DES. RNA was extracted from the liver using the trizol. SOD and GST gene expression was assed by real time RT‐PCR. The Folch Method was used to extract lipids from the liver and lipid peroxidation was determined by PeroxiDirect Kit, (Sigma). Animals treated with DES resulted in lipid peroxide formation of 14.5 nmoles/gram of tissue. Co‐administration of DAS and DES attenuated DES‐induced lipid peroxidation by 90%. No lipid peroxides were detected in the control and the DAS treated animals. DES, DAS and a combination of DES and DAS resulted in increases of SOD expression by 2.2, 10.1 and 18.1 fold respectively. Similar results were seen with GST. DES, DAS, and a combination of DES and DAS resulted in GST expression of 0.78, 8.1, and 13.2 fold respectively. Our results demonstrate that DAS inhibits DES induce lipid peroxidation. These data suggest that diallyl sulfide may prevent DES induced cancer and be used to develop potent chemopreventive compounds. Supported in part by NIH grantsRR08111 and RR03020.

Effects of fenofibrate on gene expression of carnitine palmitoyltransferase 1 in liver and skeletal muscle and its influence on insulin sensitivity

To study the effects of fenofibrate (FF), a peroxisome proliferator activated receptor (PPAR) alpha activator, on the expression of carnitine palmitoyltransferase 1 (CPT-1) mRNA in liver and muscle and its influence on insulin sensitivity. Thirty-two normal 8 week-old male SD rats were randomly divided into 3 groups: normal control group, fed with normal food for 3 weeks (NC group, n = 10), high fat diet group, fed with high fat food (HF group, n = 10), and high fat diet supplemented with FF group, fed with high fat food and given with gastric perfusion of FF (50 mg x kg(-1) x d(-1)) (FF group, n = 12). Fast serum triglyceride (TG) level was tested by automatic biochemical analyzer after 8-10 h fasting. Euglycemic-hyperinsulinemic clamp method was used to calculate the glucose infusion rate (GIR) so as to evaluate the insulin sensitivity. By the end of experiment the rats were killed with their internal organs taken out. The triglyceride (TG) contents of liver and skeletal muscle were measured using Folch method. Real-time PCR was used to detect the mRNA expression. of CPT-1 in the liver and skeletal muscles. As compared with the NC group, the TG levels of serum, liver, and skeletal muscle of the HF group were higher than those of the NC group by 0.45-fold, 2.14-fold, and 10.64-fold respectively. The GIR of the HF group was (6.2 +/- 0.8) mg x kg(-1) x min(-1), significantly lower than that of the NC group (15.8 +/- 2.1) mg x kg(-1) x min(-1), (P < 0.01), and that of the P < 0.01. The CPT-1 mRNA expression in liver of the HF group was not significantly different from that of the NC group (P > 0.05); the expression of CPT-1 mRNA in skeletal muscle of the HF group was lower than that of the NC group by 71% (P < 0.01). The CPT-1 mRNA expression in liver and skeletal muscle of the FF group were significantly higher than those of the HF group by 1.00 and 1.05 times respectively (both P < 05). The GIR was negatively correlated with the levels in the liver (r = -0.87, P < 0.01) and in the skeletal muscle (r = -0.78, P < 0.01). Fenofibrate promotes the oxygenation of fatty acids by up-regulating the CPT-1 mRNA expression in the liver and skeletal muscles, thus improving the insulin sensitivity.