A first-in-human phase I/Ib dose-escalation clinical trial of the autophagy inducer ABTL0812 in patients with advanced solid tumours.

BACKGROUND: ABTL0812 is an autophagy inducer that promotes cancer cell death by activation of cytotoxic autophagy selectively in tumour cells. ABTL0812 induces endoplasmic reticulum stress and blocks the Akt-mTOR axis; both actions converge to activate a robust and sustained autophagy leading to cancer cell death. Preclinical data supported the initiation of clinical trials in patients with cancer. PATIENTS AND METHODS: This first-in-human trial consisted of an escalation phase (3 + 3 design), followed by an expansion phase, to assess safety and tolerability of ABTL0812. Secondary objectives were determining the recommended phase II dose (RP2D), clinical antitumour activity, pharmacokinetics (PK) and pharmacodynamics (PD). RESULTS: A total of 29 patients were enrolled and treated; fifteen patients were treated in four escalation dosing cohorts (ranging from 500 mg once a day to 2000 mg twice a day) and fourteen in the expansion phase (dosed with 1300 mg three times a day). No maximum tolerated dose was attained, and RP2D was determined by PK/PD modelling. Most drug-related adverse events were gastrointestinal grade I-II. Correlation between drug levels and pAkt/Akt ratio was found. Two cases of long-term (>1 year) stable disease were observed. CONCLUSIONS: ABTL0812 is safe and has an acceptable tolerability profile, allowing a long-term oral dosing. RP2D of 1300 mg three times a day was determined according to PK/PD modelling, and preliminary antitumour efficacy was observed. CLINICAL TRIAL REGISTRATION NUMBER: NCT02201823.

N-Benzyl-linoleamide, a Constituent of Lepidium meyenii (Maca), Is an Orally Bioavailable Soluble Epoxide Hydrolase Inhibitor That Alleviates Inflammatory Pain.

Lepidium meyenii (maca), a plant indigenous to the Peruvian Andes, recently has been utilized globally for claimed health or recreational benefits. The search for natural products that inhibit soluble epoxide hydrolase (sEH), with therapeutically relevant potencies and concentrations, led to the present study on bioactive amide secondary metabolites found in L. meyenii, the macamides. Based on known and suspected macamides, 19 possible macamides were synthesized and characterized. The majority of these amides displayed excellent inhibitory potency (IC50 ≈ 20-300 nM) toward the recombinant mouse, rat, and human sEH. Quantitative analysis of commercial maca products revealed that certain products contain known macamides (1-5, 8-12) at therapeutically relevant total concentrations (≥3.29 mg/g of root), while the inhibitory potency of L. meyenii extracts directly correlates with the sum of concentration/IC50 ratios of macamides present. Considering both its in vitro efficacy and high abundance in commercial products, N-benzyl-linoleamide (4) was identified as a particularly relevant macamide that can be utilized for in vivo studies. Following oral administration in the rat, compound 4 not only displayed acceptable pharmacokinetic characteristics but effectively reduced lipopolysaccharide-induced inflammatory pain. Inhibition of sEH by macamides provides a plausible biological mechanism of action to account for several beneficial effects previously observed with L. meyenii treatments.

Species differences in toxicokinetic parameters of glycidol after a single dose of glycidol or glycidol linoleate in rats and monkeys.

Glycidol fatty acid esters (GEs) have been identified as contaminants in refined edible oils. Although the possible release of glycidol (G) from GEs is a concern, little is known about the conversion of GEs to G in the human body. This study addressed the toxicokinetics of glycidol linoleate (GL) and G in male Crl:CD(SD) rats and cynomolgus monkeys. Equimolar amounts of GL (341 mg/kg) or G (75 mg/kg) were administered by gavage to each animal. G was found in both species after the G and GL administration, while plasma GL concentrations were below the lower limit of quantification (5 ng/ml) in both species. In rats, the administration of GL or G produced similar concentration-time profiles for G. In monkeys, the C(max) and AUC values after GL administration were significantly lower than those after G administration. The oral bioavailability of G in monkeys (34.3%) was remarkably lower than that in rats (68.8%) at 75 mg/kg G administration. In addition, plasma G concentrations after oral administration at three lower doses of GL or G were measured in both species. In monkeys, G was detected only at the highest dose of G. In contrast, the rats exhibited similar plasma G concentration-time profiles after GL or G administration with significantly higher G levels than those in monkeys. In conclusion, these results indicate that there are remarkable species differences in the toxicokinetics of GEs and G between rodents and primates, findings that should be considered when assessing the human risk of GEs.

Double blind randomized clinical trial controlled by placebo with an alphalinoleic acid and prebiotic enriched cookie on risk cardiovascular factor in obese patients / Ensayo clínico aletorizado doble ciego controlado con placebo con una galleta enriquecida en ácido alfa linoleico y prebióticos en el patrón de riesgo cardiovascular de pacientes obesos

Introduction: Inulin and FOS are prebiotics with potential benefit in cardiovascular risk factors. Alphalinolenic acid (ALA) is the metabolic precursor of the long chain n-3 fatty acid eicosapentaenoic acid (20: 5n-3),this fatty acid has anti-inflammatory properties. The aim of our study was to evaluate the response of the cardiovascular risk profile in obese patients after inclusion in the diet of an ALA, FOS and inulin enriched-cookie. Material and methods: 36 patients were randomized in both branches: group I (inulin, FOS and ALA enriched cookie) Gullon SL® and group II (control cookie). Previous and after 1 month of the treatment, a nutritional and biochemical study was realized. Results: 15 patients finished the procotol in each group. In group I, a significantly increase in soluble fiber (2.3 ±0.8 g/day vs 7.7 ± 0.8 g/day: p < 0.05) and ALA (0.6 ± 0.5g/day vs 3.8 ± 0.5 g/day; p < 0.05) intakes was detected. In this group a significant decrease of total cholesterol (238.1± 45.3 mg/dl vs 210.5 ± 38.1 mg/dl: p < 0.05), LDL cholesterol(153.6 ± 23.2 mg/dl vs 127.1 ± 27.9 mg/dl: p < 0.05)and C reactive protein (6.6 ± 1.4 mg/dl vs 4.4+7-1.8 mg/dl:p < 0.05) was reached in males. Anthropometric parameters did not change in both groups. The increase in soluble fiber and ALA dietary intakes did not produce any gastrointestinal adverse effect. Conclusion: The increase of 2 grams per day of inulin,3.1 grams per day of FOS and 3.2 grams per day of alphalinolenic (ALA) dietary intakes from an enriched-cookie, improved total cholesterol, LDL cholesterol and C reactive protein levels in obese males. As far as we know, this is the first study that has evaluated the effect on risk factors of an ALA enriched cookies (AU)

Introducción: La inulina y los FOS son prebióticos con potenciales efectos beneficiosos a nivel cardiovascular. El acido alfa linolénico (ALA) es el precursor del ácido eicosapentaenoico (20: 5n-3), presentando propiedades antinflamatorias. El objetivo de nuestro trabajo es evaluar la respuesta del perfil de riesgo cardiovascular en pacientes obesos tras la inclusión en la dieta de una galleta enriquecida en inulina, FOS y ALA. Material y métodos: Un total de 36 pacientes fueron randomizados a una de las siguientes ramas: galleta I(enriquecida con inulina, FOS y ALA) y galleta II (galleta control) (Gullón SL). Cada paciente recibió un total de 2 galletas al día (70 gramos de producto). Antes de iniciar el tratamiento y al mes, se realizó una valoración nutricional y analítica. Resultados: Finalizaron el protocolo un total de 15 pacientes en cada grupo. En el grupo I se produjo un aumento significativo en la ingesta de fibra soluble (2,3 ±0,8 g/día vs 7,7 ± 0,8 g/día: p < 0,05) (inulina y FOS), así como ALA (0,6 ± 0,5 g/día vs 3,8 ± 0,5 g/día; p < 0,05). Se detectó en los pacientes varones que recibieron las galletas enriquecidas una disminución significativa de los niveles de colesterol total (238,1 ± 45,3 mg/dl vs 210,5 ±38,1 mg/dl: p < 0,05), LDL colesterol (153,6 ± 23,2 mg/dlvs 127,1 ± 27,9 mg/dl: p < 0,05) y proteína C reactiva (6,6 ±1,4 mg/dl vs 4,4+7-1,8 mg/dl: p < 0,05). No existieron diferencias estadísticamente significativas en las variables antropométricas. El aumento de la ingesta dietética de fibra soluble y ALA en los pacientes de grupo I no supuso ningún efecto secundario a nivel gastrointestinal. Conclusión: El aumento en la ingesta con una galleta enriquecida de 2 gramos al día de inulina, 3,1 gramos de FOS y 3,2 gramos de ALA, mejora en los pacientes obesos varones los niveles de colesterol total, LDL colesterol y proteína C reactiva (AU)

A novel cationic lipophosphoramide with diunsaturated lipid chains: synthesis, physicochemical properties, and transfection activities.

Cationic lipophosphoramidates constitute a class of cationic lipids we have previously reported to be efficient for gene transfection. Here, we synthesized and studied a novel lipophosphoramidate derivative characterized by an arsonium headgroup linked, via a phosphoramidate linker, to an unconventional lipidic moiety consisting of two diunsaturated linoleic chains. Physicochemical studies allowed us to comparatively evaluate the specific fluidity and fusogenicity properties of the liposomes formed. Although corresponding lipoplexes exhibited significant but relatively modest in vitro transfection efficiencies, they showed a remarkably efficient and reproducible ability to transfect mouse lung, with in vivo transfection levels higher than those observed with a monounsaturated analogue previously described. Thus, these results demonstrate that this diunsaturated cationic lipophosphoramidate constitutes an efficient and versatile nonviral vector for gene transfection. They also invite further evaluations of the transfection activity, especially in vivo, of gene delivery systems incorporating the lipid reported herein and/or other lipids bearing polyunsaturated chains.

Kinetic study of the prooxidant effect of alpha-tocopherol. Hydrogen abstraction from lipids by alpha-tocopheroxyl radical.

A kinetic study of the prooxidant effect of alpha-tocopherol was performed. The rates of allylic hydrogen abstraction from various unsaturated fatty acid esters (ethyl stearate 1, ethyl oleate 2, ethyl linoleate 3, ethyl linolenate 4, and ethyl arachidonate 5) by alpha-tocopheroxyl radical in toluene were determined, using a double-mixing stopped-flow spectrophotometer. The second-order rate constants (k (p)) obtained are <1 x 10(-2) M(-1 )s(-1) for 1, 1.90 x 10(-2) M(-1 )s(-1) for 2, 8.33 x 10(-2 )M(-1 )s(-1) for 3, 1.92 x 10(-1) M(-1 )s(-1) for 4, and 2.43 x 10(-1 )M(-1 )s(-1) for 5 at 25.0 degrees C. Fatty acid esters 3, 4, and 5 contain two, four, and six -CH(2)- hydrogen atoms activated by two pi-electron systems (-C=C-CH(2)-C=C-). On the other hand, fatty acid ester 2 has four -CH(2)- hydrogen atoms activated by a single pi-electron system (-CH(2)-C=C-CH(2)-). Thus, the rate constants, k (abstr)/H, given on an available hydrogen basis are k (p)/4 = 4.75 x 10(-3 )M(-1 )s(-1) for 2, k (p)/2 = 4.16 x 10(-2) M(-1 )s(-1) for 3, k (p)/4 = 4.79 x 10(-2 )M(-1 )s(-1) for 4, and k (p)/6 = 4.05 x 10(-2 )M(-1 )s(-1) for 5. The k (abstr)/H values obtained for 3, 4, and 5 are similar to each other, and are by about one order of magnitude higher than that for 2. From these results, it is suggested that the prooxidant effect of alpha-tocopherol in edible oils, fats, and low-density lipoproteins may be induced by the above hydrogen abstraction reaction.

Amended safety assessment of tall oil acid, sodium tallate, potassium tallate, and ammonium tallate.

Tall oil acid is a mixture of oleic and linoleic acids (fatty acids) and rosin acids derived from tall oil, a by-product of pulp from resinous woods, used in cosmetic products as a surfactant at concentrations up to 8%. Ammonium, potassium, and sodium salts also are listed as cosmetic ingredients. In addition to the studies summarized in this report, extensive toxicity, genotoxicity, and carcinogenicity studies in animals are available for oleic, lauric, palmitic, myristic, and stearic fatty acids as published earlier by the Cosmetic Ingredient Review (CIR). These data may be extrapolated to tall oil acid and its salts. There are no reports of current uses or use concentration data for ammonium tallate, nor are use concentration data available for the other salts. The CIR Expert Panel found tall oil acid, ammonium tallate, potassium tallate, and sodium tallate to be safe cosmetic ingredients in the given practices of use and concentration.

Incorporation of cis-9,trans-11 or trans-10,cis-12 conjugated linoleic acid into plasma and cellular lipids in healthy men.

This study investigated the incorporation of cis-9,trans-11 conjugated linoleic acid (c9,t11 CLA) and trans-10,cis-12-CLA (t10,c12 CLA) into plasma and peripheral blood mononuclear cell (PBMC) lipids when consumed as supplements highly enriched in these isomers. Healthy men (n = 49, age 31 +/- 8 years) consumed one, two, and four capsules containing approximately 600 mg of either c9,t11 CLA or t10,c12 CLA per capsule for sequential 8 week periods followed by a 6 week washout before consuming the alternative isomer. Both isomers were incorporated in a dose-dependent manner into plasma phosphatidylcholine (PC) (c9,t11 CLA r = 0.779, t10,c12 CLA r = 0.738; P < 0.0001) and cholesteryl ester (CE) (c9,t11 CLA r = 0.706, t10,c12 CLA r = 0.788; P < 0.0001). Only t10,c12 CLA was enriched in plasma nonesterified fatty acids. Both c9,t11 CLA and t10,c12 CLA were incorporated linearly into PBMC total lipids (r = 0.285 and r = 0.273, respectively; P < 0.0005). The highest concentrations of c9,t11 CLA and t10,c12 CLA in PBMC lipids were 3- to 4-fold lower than those in plasma PC and CE. These data suggest that the level of intake is a major determinant of plasma and PBMC CLA content, although PBMCs appear to incorporate both CLA isomers less readily.

Growth and fatty acid metabolism of human breast cancer (MCF-7) xenografts in nude rats: impact of constant light-induced nocturnal melatonin suppression.

The nocturnal melatonin (MLT) surge is a relevant oncostatic signal for a variety of experimental malignancies. Population studies support the hypothesis that exposure to light at night may represent a new risk factor for breast cancer possibly through the suppression of pineal MLT production and/or circadian disruption. We tested the ability of constant light exposure to suppress MLT production in female nude rats and stimulate the growth of tissue-isolated MCF-7 human breast cancer xenografts via increased tumor linoleic acid (LA) metabolism. Rats maintained on an alternating light/dark cycle (L:D group) exhibited a robust circadian MLT rhythm that was abolished following constant light exposure. During the exposure of animals bearing tissue-isolated human MCF-7 breast cancer xenografts to constant light, the rate of tumor growth markedly increased relative to the L:D group. Tumor LA uptake and its metabolism to the mitogen 13-hydroxyoctadecadienoic acid (13-HODE) were also substantially higher under constant light conditions. This is the first biological evidence for a potential link between constant light exposure and increased human breast oncogenesis involving MLT suppression and stimulation of tumor LA metabolism.

Preferential incorporation of trans, trans-conjugated linoleic acid isomers into the liver of suckling rats.

The present study was designed to compare the conjugated linoleic acid (CLA) isomeric distribution pattern in the liver of suckling rats in relation to those in the milk and maternal diet. Silver-ion HPLC was used to separate individual CLA isomers. It was found that the isomeric distribution pattern in the milk was very similar to that in the maternal dietary fat. However, the CLA isomeric distribution patterns in the liver phospholipids (PL) and triacylglycerols were different from those in the diet and milk. In the liver PL, total cisltrans isomers accounted for 63.6-63.9% of total CLA, which was in contrast to the values of 88.1-89.1% in the milk and diet. In the liver PL, total transltrans isomers were 20.6-20.8% of the total CLA isomers whereas they were only 2.6-3.7% in the milk and diet. It is concluded that trans/trans-CLA were preferentially incorporated into the liver whereas for the incorporation of cis/trans-CLA there was partial discrimination.

Perturbation of lipid metabolism by linoleic acid hydroperoxide in CaCo-2 cells.

Dietary hydroperoxides are being discussed as potential health hazards contributing to oxidative stress-related diseases. However, how food-born hydroperoxides could exert systemic effects remains elusive in view of the limited chances to be absorbed. Therefore, the metabolic fate of 13-HPODE (13-hydroperoxy octadecadienoic acid), 13-HODE (13-hydroxy octadecadienoic acid) and linoleic acid (LA) was investigated in a CaCo-2 cell monolayer as a model of the intestinal epithelium. [1-14C]-13-HPODE, up to a non-cytotoxic concentration of 100 microM, did not cross the CaCo-2 cell monolayer unreduced if applied to the luminal side. The [1 -14C]-HPODE-derived radioactivity was preferentially recovered from intracellular and released diacylglycerols (DG), phospholipids (PL) and cholesterol esterified with oxidized fatty acids (oxCE). A similar distribution pattern was obtained with 13-HODE. In contrast, LA is preferentially incorporated into triacylglycerols (TG), cholesteryl esters (CE) and PL (but mainly released as TG). 13-HPODE dose-dependently decreased the incorporation of LA into released TG, while LA accumulated in cellular and released DGs, effects similarily exerted by 13-HODE. We concluded that food-born hydroperoxy fatty acids are instantly reduced by the gastrointestinal glutathione peroxidase, which was previously shown to persist in selenium deficiency. Accordingly, modulation of the glutathione peroxidases by selenium deprivation/repletion did not modify the disturbance of the lipid metabolism by 13-HPODE. Thus, hydroperoxy fatty acids disturb intestinal lipid metabolism by being esterified as hydroxy fatty acids into complex lipids, and may render lipoproteins synthesized thereof susceptible to further oxidative modifications.

Conjugated linoleic acids: all the same or to everyone its own function?

Conjugated linoleic acid (CLA) is a generic term referring to a mixture of geometrical and positional isomers of linoleic acid in which up to 16 members have been identified. Many potentially beneficial health effects have been ascribed to these fatty acids when consumed as a mixture, and where generally 2 isomers dominate, e.g. the 9c, 11t-isomer, the so-called rumenic acid, and the 10t, 12c-isomer: anti-carcinogenic, immune modulator, anti-atherosclerotic, and anti-obesity among the most spectacular. The question arises as to whether the pleiotropic biological activity is supported by one or several of the isomers. Recent studies using pure individual isomers have started to elucidate this issue, but many others are required to ascribe a respective role to each CLA isomer (the main ones as well as the minor ones), such as those occurring in some complex mixtures already commercially available, or even in foodstuff. The aim of the present study was to focus on the CLA-isomer specific effects depicted in the literature up to now.

Milk fat conjugated linoleic acid (CLA) inhibits growth of human mammary MCF-7 cancer cells.

The relationship between growth and the antioxidant enzyme defence system in human MCF-7 (breast) cancer cells treated with bovine milk fat enriched with conjugated linoleic acid (CLA) was studied. Milk enriched in CLA was obtained from cows on pasture supplemented with full fat rapeseeds and full fat soyabeans (1). Cell number decreased up to 90% (p < 0.05) and lipid peroxidation increased 15-fold (p < 0.05) following incubation of MCF-7 cells for 8 days with increasing levels of milk fat yielding CLA concentrations between 16.9 and 22.6 ppm. Growth suppression and prooxidant effects of milk fat CLA were independent of the variable composition of the milk fat samples, suggesting that CLA was the active ingredient in milk fat responsible for the cytotoxic effect. Mixtures containing isomers of CLA (c9, t11-, t10, c12-, c11, t13- and minor amounts of other isomers) and linoleic acid (LA) at similar concentrations to the milk fat samples were as effective at inhibiting growth and stimulating peroxidation of MCF-7 cells as the milk fatty acids. Incubation of the cells with the c9, t11 CLA isomer (20 ppm) or the mixture of CLA isomers (20 ppm) for 8 days resulted in a 60% decrease (p < 0.05) in viability compared with untreated controls and was significantly (p < 0.05) more effective than incubation with the t10, c12 CLA isomer (20 ppm), which caused only a 15% decrease in cell numbers under similar conditions. A 25% increase (p < 0.05) in cell proliferation occurred when LA (20 ppm) alone was incubated with MCF-7 cells for 8 days. 14C-CLA was preferentially incorporated into the phospholipid fraction of the MCF-7 cell lipids in a dose-dependent manner and CLA accumulated in cell membranes more efficiently when the cells were incubated in the presence of milk fat than the c9, t11 synthetic CLA isomer. Superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activities were induced in MCF-7 cells exposed to milk fat (containing 16.9-22.6 ppm CLA) over 8 days. The data indicate that milk fat triglyceride-bound CLA, consisting primarily of the c9, t11 isomer, was cytotoxic towards MCF-7 cells.

Gastrointestinal glutathione peroxidase prevents transport of lipid hydroperoxides in CaCo-2 cells.

BACKGROUND & AIMS: Gastrointestinal glutathione peroxidase (GI-GPx), 1 of the 4 types of selenium-dependent glutathione peroxidases, is expressed exclusively in the gastrointestinal system and has therefore been suggested to function as a barrier against the absorption of dietary hydroperoxides. METHODS: The selenium-dependent expression of GI-GPx and cytosolic GPx (cGPx) was analyzed by Western blotting. Transport of 13-hydroperoxy octadecadienoic acid (13-HPODE) was investigated in a CaCo-2 cell monolayer modulated in GI-GPx and cGPx by selenium restriction or repletion. Localization of GI-GPx in rat intestine was visualized by immunohistochemistry. RESULTS: Low but significant GI-GPx levels were detected in selenium-deficient CaCo-2 cells and in the gastrointestinal tract of selenium-deficient rats, whereas cGPx was completely absent. Selenium supplementation of CaCo-2 cells resulted in a 5-fold increase of GI-GPx protein, whereas total GPx activity increased by a factor of 13, with most of the GPx activity under selenium-adequate conditions being cGPx. Irrespective of the selenium status, 13-HPODE did not reach the basolateral side of an intact CaCo-2 cell monolayer. Depending on the selenium status, hydroperoxides damaged the monolayer as evidenced by loss of transepithelial resistance and paracellular diffusion of lucifer yellow. Only under these conditions was unmetabolized 13-HPODE detectable at the basolateral side. CONCLUSIONS: Low GI-GPx levels, as present in selenium deficiency, suffice to prevent transport of 13-HPODE. GI-GPx may thus function as a barrier against hydroperoxide absorption. cGPx contributes to balance major oxidative challenge.

Lymphatic delivery and in vitro pancreatic lipase hydrolysis of glycerol esters of conjugated linoleic acids in rats.

We examined the intestinal delivery of conjugated linoleic acids (CLA) given in their triacylglycerol form in the mesenteric lymph of rats. Emulsions containing a mixture of the trilinolein/triester of CLA (9:1) and a tri-[1-(14)C]-linoleyl-sn-glycerol tracer were administered by force-feeding. Lymph was collected over two time periods (0-6 and 6-24 h), and the apparent recovery of CLA was determined relative to that of [1-(14)C]-18:2(n-6). A mixture of CLA-triester/trilinolein (1:9), trilinolein or CLA-triester was separately subjected to pancreatic lipase hydrolysis in vitro to determine whether the lymphatic recovery of CLA was correlated with the initial step of digestion. Lymphatic recovery of CLA was similar to that of 18:2(n-6) (95.6+/-9.0% of the linoleic acid recovery), and isomer repartition was similar in lymph and in the oil fed, indicating that all the CLA isomers were equally absorbed by the enterocytes. Unexpectedly, the in vitro release of CLA into the absorbable forms (free fatty acids and 2-monoacyl-sn-glycerol) was consistently lower than that of 18:2(n-6). Moreover, the 9c, 11t-isomer of CLA was also released faster into the absorbable forms than its 10t,12c homolog (P = 0.05). We cannot ascribe a distinct cellular accumulation or a difference in the biological effects of different CLA isomers on the ground of a selective intestinal absorbability. Also, the physiological conditions prevailing in vivo in the digestive tract are likely to overcome the relative resistance of CLA ester bonds to pancreatic lipase hydrolysis and allow a lymphatic recovery of CLA similar to that of linoleic acid.

Effect of dietary docosahexaenoic acid on desaturation and uptake in vivo of isotope-labeled oleic, linoleic, and linolenic acids by male subjects.

The effect of dietary docosahexaenoic acid (22:6n-3, DHA) on the metabolism of oleic, linoleic, and linolenic acids was investigated in male subjects (n = 6) confined to a metabolic unit and fed diets containing 6.5 or <0.1 g/d of DHA for 90 d. At the end of the diet period, the subjects were fed a mixture of deuterated triglycerides containing 18:1n-9[d6], 18:2n-6[d2], and 18:3n-3[d4]. Blood samples were drawn at 0, 2, 4, 6, 8, 12, 24, 48, and 72 h. Methyl esters of plasma total lipids, triglycerides, phospholipids, and cholesterol esters were analyzed by gas chromatography-mass spectrometry. Chylomicron triglyceride results show that the deuterated fatty acids were equally well absorbed and diet did not influence absorption. Compared to the low-DHA diet (LO-DHA), clearance of the labeled fatty acids from chylomicron triglycerides was modestly higher for subjects fed the high DHA diet (HI-DHA). DHA supplementation significantly reduced the concentrations of most n-6[d2] and n-3[d4] long-chain fatty acid (LCFA) metabolites in plasma lipids. Accumulation of 20:5n-3[d4] and 22:6n-3[d4] was depressed by 76 and 88%, respectively. Accumulations of 20:3n-6[d2] and 20:4n-6[d2] were both decreased by 72%. No effect of diet was observed on acyltransferase selectivity or on uptake and clearance of 18:1n-9[d6], 18:2n-6[d2], and 18:3n-3[d4]. The results indicate that accumulation of n-3 LCFA metabolites synthesized from 18:3n-3 in typical U.S. diets would be reduced from about 120 to 30 mg/d by supplementation with 6.5 g/d of DHA. Accumulation of n-6 LCFA metabolites synthesized from 18:2n-6 in U.S. diets is estimated to be reduced from about 800 to 180 mg/d. This decrease is two to three times the amount of n-6 LCFA in a typical U.S. diet. These results support the hypothesis that health benefits associated with DHA supplementation are the combined result of reduced accretion of n-6 LCFA metabolites and an increase in n-3 LCFA levels in tissue lipids.

Distributions of conjugated linoleic acid (CLA) isomers in tissue lipid classes of pigs fed a commercial CLA mixture determined by gas chromatography and silver ion-high-performance liquid chromatography.

Pigs were fed a commercial conjugated linoleic acid (CLA) mixture, prepared by alkali isomerization of sunflower oil, at 2% of the basal diet, from 61.5 to 106 kg live weight, and were compared to pigs fed the same basal diet with 2% added sunflower oil. The total lipids from liver, heart, inner back fat, and omental fat of pigs fed the CLA diet were analyzed for the incorporation of CLA isomers into all the tissue lipid classes. A total of 10 lipid classes were isolated by three-directional thin-layer chromatography and analyzed by gas chromatography (GC) on long capillary columns and by silver-ion high-performance liquid chromatography (Ag+-HPLC); cholesterol was determined spectrophotometrically. Only trace amounts (<0.1%; by GC) of the 9,11-18:2 cis/trans and trans,trans isomers were observed in pigs fed the control diet. Ten and twelve CLA isomers in the diet and in pig tissue lipids were separated by GC and Ag+- HPLC, respectively. The relative concentration of all the CLA isomers in the different lipid classes ranged from 1 to 6% of the total fatty acids. The four major cis/trans isomers (18.9% 11 cis,13 trans-18:2; 26.3% 10 trans,12 cis-18:2; 20.4% 9 cis,11 trans-18:2; and 16.1% 8 trans, 10 cis-18:2) constituted 82% of the total CLA isomers in the dietary CLA mixture, and smaller amounts of the corresponding cis,cis (7.4%) and trans,trans (10.1%) isomers were present. The distribution of CLA isomers in inner back fat and in omental fat of the pigs was similar to that found in the diet. The liver triacylglycerols (TAG), free fatty acids (FFA), and cholesteryl esters showed a similar pattern to that found in the diet. The major liver phospholipids showed a marked increase of 9 cis,11 trans-18:2, ranging from 36 to 54%, compared to that present in the diet. However, liver diphosphatidylglycerol (DPG) showed a high incorporation of the 11 cis,13 trans-18:2 isomer (43%). All heart lipid classes, except TAG, showed a high content of 11 cis,13 trans-18:2, which was in marked contrast to results in the liver. The relative proportion of 11 cis,13 trans-18:2 ranged from 30% in the FFA to 77% in DPG. The second major isomer in all heart lipids was 9 cis,11 trans-18:2. In both liver and heart lipids the relative proportions of both 10 trans,12 cis-18:2 and 8 trans, 10 cis-18:2 were significantly lower compared to that found in the diet. The FFA in liver and heart showed the highest content of trans,trans isomers (31 to 36%) among all the lipid classes. The preferential accumulation of the 11 cis,13 trans-18:2 into cardiac lipids, and in particular the major phospholipid in the inner mitochondrial membrane, DPG, in both heart and liver, appears unique and may be of concern. The levels of 11 cis,13 trans-18:2 naturally found in foods have not been established.

Permeability and metabolic properties of a trophoblast cell line (HRP-1) derived from normal rat placenta.

The HRP-1 cell line is derived from normal rat placenta and appears morphologically similar to and retains characteristic expression of cellular markers of labyrinthine trophoblast cells. In this study, monolayers of HRP-1 cells grown on permeable supports were evaluated as a potential in vitro system to study trophoblast transport and metabolism. The cell line was shown to express and retain functional activity of the predominant placental cytochrome P450 isozyme, CYP1A1. Additionally, the HRP-1 cells retain functional activity of angiotensin I converting enzyme and carboxypeptidase N-like enzyme, peptidases characteristic of the trophoblast. The permeation of several hydrophilic, inert markers across the HRP-1 monolayers was observed to be dependent on effective molecular size and to be passive in nature. Functional asymmetry of the HRP-1 cells was illustrated by the predominant permeation of linoleic acid in the apical-to-basolateral direction across the monolayers. Transferrin passage across HRP-1 monolayers was concentration-dependent, was bidirectional, and could be inhibited by unlabeled transferrin, features typical of the trophoblast transport system for transferrin. Collectively, these properties suggest that the HRP-1 cell line may provide a useful tool for evaluating some of the permeability and metabolic properties of the trophoblast.

Dietary linoleic acid intake controls the arterial blood plasma concentration and the rates of growth and linoleic acid uptake and metabolism in hepatoma 7288CTC in Buffalo rats.

In this study, we tested the hypothesis that dietary linoleic acid intake controls the arterial blood plasma linoleic acid concentration and the rates of tumor growth and linoleic acid metabolism in vivo. Seven groups of young male Buffalo rats (11-21 rats/group) were given free access to semipurified diets containing different amounts of corn and/or olive oils. Four other groups (7-11 rats/group) were 30% energy-restricted. Each experiment included periods for rat growth and plasma lipid stabilization (6 wk), measurement of mean daily arterial blood plasma fatty acid concentrations (3 wk), surgical implantation of a subcutaneous tissue-isolated hepatoma 7288CTC, tumor growth and harvest (2-4 wk). Linoleic + arachidonic acid (P = 0.007) and oleic acid (P = 0.002) concentrations in arterial blood plasma were increased as dietary intake of linoleic and oleic acids was increased, respectively. In rats given free access to food, tumor growth was directly dependent on the plasma concentrations of linoleic (P < 0.001) and arachidonic acids (P = 0.04). Tumor growth in energy-restricted rats was dependent only on the linoleic acid concentration (P = 0.008). Energy restriction itself caused a growth inhibition independent of plasma linoleic acid. The linoleic acid and total fatty acid concentrations of tumor triacylglycerols were directly dependent on the plasma linoleic acid concentration in rats given free access to food (P = 0.009). Hepatoma 7288CTC (both in vivo and during perfusion in situ) supported a dose-dependent conversion (P < 0.001) of plasma linoleic acid to the mitogen, 13-hydroxy-9, 11-octadecadienoic acid. We conclude that increased arterial blood plasma linoleic acid concentrations, caused by increased dietary intakes, specifically stimulate growth, lipid storage and linoleic acid metabolism in hepatoma 7288CTC in vivo.

Nutrient uptake into undifferentiated and differentiated HT-29 cells in culture.

HT-29 human colon carcinoma cells in culture have many characteristics of enterocytes, and these cells have been used by others to study intestinal drug and nutrient transport and metabolism. When grown in glucose-containing medium, HT-29 cells are largely undifferentiated (HT-29glu), but when grown in the absence of glucose but in the presence of galactose (HT-29gal), the population of cells is mostly differentiated. This study was undertaken with HT-29glu and HT-29gal cells to study the uptake of palmitic acid (16:0), linoleic acid (18:2), and cholesterol. The relationship between concentration and uptake of 16:0, 18:2, and cholesterol was linear in HT-29glu and HT-29gal cells, with the relative values of the slopes of this relationship being 18:2 > > 16:0 > > cholesterol. The rates of uptake of these lipids were at least three times higher in HT-29gal than in HT-29glu cells. In HT-29glu cells, the relative rates of uptake of the sugars at 32 mM were D-glucose = galactose > fructose > > alpha-methylglucose. Uptake of these sugars was much greater in HT-29gal than in HT-29glu cells. When 100 microM forskolin was added to the incubation medium for 7 days post-confluency, which stimulates the activity of adenylate cyclase and thereby increases the intracellular synthesis of cAMP, there was no effect on the uptake of the lipids or the sugars in either HT-29glu or HT-29gal cells. Thus, (i) differentiated HT-29gal cells transport larger amounts of lipids and sugars than do undifferentiated HT-29glu cells; (ii) forskolin has no effect on the uptake of lipids or sugars in these cells. This human cell culture system may be useful to study the in vitro transport of lipids, to establish the role of cell differentiation on these uptake processes, and to determine the potential role of selected intracellular signals.