Control of cyclic AMP concentration in bovine endometrial stromal cells by arachidonic acid

Second messenger signalling through cyclic AMP (cAMP) plays an important role in the response of the endometrium to prostaglandin (PG) E(2) during early pregnancy. Arachidonic acid, which is a by-product of the luteolytic cascade in ruminants, is a potential paracrine signal from the epithelium to the stroma. We investigated the effects of arachidonic acid on the response of the stroma to PGE(2). cAMP was measured in bovine endometrial stromal cells treated with agents known to activate or inhibit adenylyl cyclase, protein kinase C (PKC) or phosphodiesterase (PDE). PGE(2) increased the intracellular cAMP concentration within 10 min, and this effect was attenuated by arachidonic acid and the PKC activator, 4beta-phorbol myristate acetate (PMA). The inhibitory effect of arachidonic acid on PGE(2)-induced cAMP accumulation was prevented by the PKC inhibitor, RO318425, and was absent in cells in which PKC had been downregulated by exposure to PMA for 24 h. The effect of arachidonic acid was also prevented by the PDE inhibitor, 3-isobutyl-1-methylxanthine. Arachidonic acid was shown by immunoblotting to prevent induction of cyclooxygenase-2 by PGE(2), forskolin or dibutyryl cAMP. The results indicate that arachidonic acid activates PDE through a mechanism involving PKC, counteracting a rise in intracellular cAMP in response to PGE(2). The data suggest that arachidonic acid antagonizes PGE(2) signalling through cAMP in the bovine endometrium, possibly acting to ensure a rapid return to oestrus in the case of failure of the maternal recognition of pregnancy.     

Dietary Plant Sterols Supplementation Increases In Vivo Nitrite and Nitrate Production in Healthy Adults: A Randomized,Controlled Study

A randomized, double‐blinded, placebo‐controlled and crossover study was conducted to simultaneously measure the effects, 3 h after consumption and after 4‐wk daily exposure to plant sterols‐enriched food product, on in vivo nitrite and nitrate production in healthy adults. Eighteen healthy participants (67% female, 35.3 [mean] ± 9.5 [SD] years, mean body mass index 22.8 kg/m²) received 2 soy milk (20 g) treatments daily: placebo and one containing 2.0 g free plant sterols equivalent of their palmityl esters (β‐sitosterol, 55%; campesterol, 29%; and stigmasterol, 23%). Nitrite and nitrate concentrations were measured in the blood plasma and urine, using stable isotope‐labeled gas chromatography‐mass spectrometry. L‐arginine and asymmetric dimethylarginine concentrations in blood serum were measured using commercially available enzyme immunoassays. Nitrite and nitrate concentrations in blood plasma (nitrite 5.83 ± 0.50 vs. 4.52 ± 0.27; nitrate 15.78 ± 0.96 vs. 13.43 ± 0.81 μmol/L) and urine (nitrite 1.12 ± 0.22 vs. 0.92 ± 0.36, nitrate 12.23 ± 1.15 vs. 9.71 ± 2.04 μmol/L) were significantly elevated after 4‐wk plant sterols supplementation Placebo and 3‐h treatments did not affect the blood plasma and urinary concentrations of nitrite and nitrate. Circulating levels of L‐arginine and asymmetric dimethylarginine were unchanged in the placebo and treatment arms. Total plant sterols, β‐Sitosterol, campesterol, and stigmasterol concentrations were significantly elevated after 4‐wk treatments compared to the placebo and 3‐h treatments. Blood plasma nitrite and nitrate concentrations correlated significantly with the plasma total and specific plant sterol concentrations. Our results suggest that dietary plant sterols, in the combination used, can upregulate nitrite, and nitrate production in vivo.     

UPLC-MS法测定烤后烟叶中的5种游离态甾醇

为考察国内不同类型、品种、产地和部位烟叶样品中游离态甾醇质量分数的分布情况,采用UPLC-MS联用法测定了国内烤烟、雪茄烟和晒烟共47个烟叶样品中5种甾醇(豆甾醇、β-谷甾醇、菜油甾醇、胆甾醇、麦角甾醇)的质量分数。结果表明:(1)烟叶样品中游离态甾醇的总质量分数呈现特定规律,其中豆甾醇质量分数最高,其次是β-谷甾醇、菜油甾醇、胆甾醇和麦角甾醇;(2)烤烟和晒烟中β-谷甾醇的质量分数大于菜油甾醇,而雪茄烟中菜油甾醇的质量分数大于β-谷甾醇;(3)不同品种、部位和产地的烟叶中游离态甾醇的质量分数存在差异。     

Total Plant Analyses of Sterols and Fatty Acids of the Winged Bean (Psophocarpus tetragonolobus)

Various parts of the winged bean plant, Psophocarpus tetragonolobus were analyzed for sterols and fatty acids. The major sterols were sitosterol, stigmasterol and campesterol. Sitosterol occurred in greatest amounts except in roots where stigmasterol was the predominant sterol. In seeds, behenic acid (22:0) comprised 13.5% of the total fatty acids, whereas, in the other plant parts it varied from 0.1–6.9%. Leaves contained the highest level of fatty acids, but only 22% of these fatty acids were saturated.     

几种常见坚果植物甾醇组成及含量测定

采用毛细管气相色谱法,对葵花籽、花生、核桃及松子中植物甾醇组成进行分析并测定其含量。结果表明：四种坚果中均含有菜油甾醇和β-谷甾醇,均未检测到菜籽甾醇及豆甾醇;其甾醇总含量以松子最高,葵花子和花生次之,最低为核桃。同时,在所有样品中测出三种甾醇中均以β-谷甾醇含量最高。     

气相色谱- 质谱法测定蜂王浆中的植物甾醇组成

为探明蜂王浆中的植物甾醇组成,蜂王浆样品经冻干、萃取得到王浆脂类物质,再经过皂化、衍生后使用HP-5MS 柱进行分离,采用GC-MS 对蜂王浆中甾醇类物质进行鉴定,并用GC-FID 内标法进行定量分析。结果表明：蜂王浆中除含有少量胆固醇外,还含有25- 羟基-24- 甲基胆甾醇、菜油甾醇、豆甾醇、β - 谷甾醇、Δ5-燕麦甾醇、Δ7- 燕麦甾醇等植物甾醇;蜂王浆中总甾醇含量在51mg/100g 鲜王浆以上。其中,25- 羟基-24- 甲基胆甾醇、菜油甾醇、β- 谷甾醇和Δ5- 燕麦甾醇含量较高,分别占总甾醇含量的36.79%、6.30%、19.75% 和27.99%。     

SEPARATION OF STEROLS AND TRITERPENE ALCOHOLS FROM UNSAPONIFIABLE FRACTIONS OF THREE PLANT SEED OILS

Preparative HPLC was used to separate sterols and triterpene alcohols from the unsaponifiable matters in plant oils from Camellia weiningensis L., Brassica juncea L. and Microula sikkimensis . The isolated sterols and triterpene alcohols were acetylated and further purified by AgNO₃ impregnated silica gel preparative thin layer chromatography (TLC). The isolated acetyl derivatives of sterols and triterpene alcohols were identified by melting point, specific rotation, infrared and mass spectrometry. The sterols were brassicasterol, campesterol, stigmasterol, β-sitosterol and Θ⁵-avenasterol, Θ⁷-avenasterol, Θ⁷-stigmastenol and α-spinasterol. The triterpene alcohols were cycloartanol, cycloartenol, 24-methylenecycloartanol, cyclobranol, dammaradienol, tirucalla-7,24-dienol, butyrospermol, β-amyrin, germanicol, α-4-taraxasterol and lupeol.     

HPLC-UV法测定植物甾醇中3种甾醇单体的含量

本文建立了一种利用紫外高效液相法(HPLC-UV)测定植物甾醇中菜油甾醇、豆甾醇和β-谷甾醇的方法。采用 Waters Symmetry C₁₈柱(4.6 mm×250 mm,5 μm),以乙腈和水为流动相等度洗脱,柱温30 ℃,流速1.0 mL/min,紫外检测波长为208 nm。实验结果表明:菜油甾醇、豆甾醇和β-谷甾醇在38 min内均能实现基线分离,线性范围分别为2.52~50.30、5.08~101.60、5.10~102.00 μg/mL时,甾醇单体的线性关系良好,相关系数r分别为0.9971、0.9989、0.9991,检测限为2.5 μg/mL;日内精密度在0.06%~3.06%范围内,日间精密度介于1.56%~6.61%;加样回收率介于92.74%~106.25%之间。本方法能够准确测定4种植物甾醇中3种甾醇单体的含量,其中大豆甾醇和木甾醇中3种甾醇单体的含量组成差异较大,大豆甾醇中菜油甾醇和豆甾醇的含量分别为163.80~251.23 mg/g和134.89~235.04 mg/g,远高于木甾醇中的菜油甾醇和豆甾醇含量,而木甾醇中β-谷甾醇含量为(685.10±7.55) mg/g,则明显高于β-谷甾醇在大豆甾醇中的含量。相对现有的高效液相方法,本方法实现了对混合甾醇中菜油甾醇、豆甾醇和β-谷甾醇3种甾醇单体的精确定量分析。     

Development of a Method for the Analysis of Sterols in Sterol-Enriched Deli-Style Turkey with GC-FID

Plant sterols have been recognised by the European Food Safety Authority for their cholesterol-lowering properties and are currently added to several food formulations. The objective of this study was to develop a method based on formation of sterol trimethylsilyl ether derivatives and separation and quantification by GC for the determination of three phytosterols (β-sitosterol, stigmasterol and campesterol) in sterol-enriched deli-style turkey. The assay was linear (concentration range 4.3–172.1 μg/ml, R ²?≥?0.9868) and accuracy and precision were within the acceptance criteria of the U.S. Food and Drug Administration (USFDA) guidelines for method validation, set at <20 % RSD at the lower limit of quantification (LLOQ) and <15 % RSD for all other standards. Accuracy measured by relative response factor (RRF) also met the USFDA validation criteria. No matrix effects were observed. The response factors (RFs) of the three sterols differed significantly to that of the internal standard (ISTD) used, leading to RRF dissimilar to 1 (campesterol?=?1.0167, stigmasterol?=?1.4458, β-sitosterol?=?0.9029). This method is suitable for quantification in meat matrix, and it has been successfully applied to the determination of sterols in sterol-enriched deli-style turkey (21 mg sterols/0.5 g sample).     

Effects of low hydrostatic pressure and moderate heat on texture,pectic substances and color of carrot

Sterols in the seeds of wild Finnish blueberry (Vaccinium myrtillus) and lingonberry (Vaccinium vitis-idaea) were analyzed as TMS derivatives by gas chromatography-mass spectrometry. Free and esterified sterols constituted 0.7% and 0.3% of the seed oil of V. myrtillus, respectively, whereas in the seed oil of V. vitis-idaea the sterols in the two fractions were equally represented at 0.5-0.6%. Sitosterol (85% in V. myrtillus and 80% in V. vitis-idaea) and campesterol (7% in V. myrtillus and 6% in V. vitis-idaea) were the dominant free sterols. In comparison to free sterols, steryl esters were found to have considerably lower proportions of sitosterol (40-60%) and campesterol (3-5%), accompanied by higher levels of intermediates in the biosynthetic cascade. Although both species contained the same major sterols, they differed in their relative abundances of individual compounds. Specifically, blueberry contained a higher proportion of sitosterol and campesterol and a lower proportion of isofucosterol and cycloartenol. Comparison of berries collected from northern and southern Finland showed that growth conditions have little effect on the sterol compositions in the berries. To our knowledge this is the first report on the content and composition of phytosterols in Vaccinium sp. The results provide important information on the chemical compositions, nutritional properties, and possible chemotaxonomical characteristics of the two species.     

Sterols and Triterpenyl alcohols in common pulpwoods and black liquor soaps

The amount and composition of lipophilic extractives, and especially of sterols and triterpenyl alcohols, were analysed for six important pulpwood species, i.e., Scots pine, Loblolly and Longleaf pine, Norway spruce, Siberian larch and Silver birch. Sulphate soaps (tall oil soaps) derived from these species were also analysed. Scots pine and Loblolly pine contained the highest amounts and Siberian larch the lowest amounts of lipophilic extractives. The highest amounts of sterols were found in birch. The sterols in wood occurred mainly as esters. Sitosterol was the main sterol in all species. Isolation and analysis of the steryl esters confirmed the natural occurrence of sitostanol esters in wood. Considering the use of sitosterol or sitostanol as cholesterol-lowering component in food products, a high ratio of sitosterol to campesterol is beneficial. This ratio was high in birch and pines but clearly lower in spruce and larch. The highest ratios of sitosterol to campesterol were found in soaps from pines and birch.     

Effect of water cooking on free phytosterol levels in beans and vegetables

Plant sterols (phytosterols) are known to decrease plasma cholesterol, mainly the atherogenic LDL cholesterol. In an earlier study, the thermal stability of phytosterols in vegetable oils was reported. The aim of this present work was to investigate the potential effect of cooking (30 min in boiling water), for eight plant products (broad bean, celery, cabbage, courgette, carrot, cauliflower, onion, pepper), on the free phytosterol level. Sitosterol was the most abundant sterol, followed by campesterol. After cooking, the level of total sterols was higher in all vegetables than that before cooking, if dry matter is considered. Acid hydrolysis (active for glycosylated phytosterols) yielded a higher sterol value than alkaline hydrolysis alone (active for esterified phytosterols). This indicated that studied vegetables contained appreciable amounts of steryl glycosides. Their cooking induced higher values of free phytosterols. Cooked vegetables could give better protection against cardiovascular diseases thanks to higher phytosterol levels.     

Potential bioactive effects of casein hydrolysates on human cultured cells

The potential bioactivity of eight distinct casein hydrolysates (designated a–h) was assessed by investigating different parameters on human cultured cells. Following 24 h supplementation, the casein hydrolysates exerted varying effects on the viability and growth of Jurkat T cells, with IC₅₀ values ranging from 19.5% to 66.8% (v/v). Treatment with the hydrolysates did not affect the membrane integrity or superoxide dismutase activity of Jurkat cells. Sample a significantly affected both cellular catalase activity and reduced glutathione (GSH) content, whereas samples c, d, and e enhanced (P < 0.05) GSH content. Neither genotoxic nor genoprotective effects were exerted by the casein hydrolysates. Interestingly, the casein hydrolysates d–h significantly increased Concanavalin A (ConA)-stimulated IL-2 levels but had no effect on ConA-induced IL-10 production in the Jurkat cells. The differing bioactive effects of these casein hydrolysates may, in part, be attributed to differences in the enzyme specificities of the enzyme activities used in their preparation.     

Short communication: Protein kinase C regulates glucose uptake and mRNA expression of glucose transporter (GLUT) 1 and GLUT8 in lactating bovine mammary epithelial cells

The aim of this study was to determine the role of protein kinase C (PKC) in regulating glucose uptake in lactating bovine mammary epithelial cells (BMEC). The BMEC were cultured and treated with different concentrations of phorbol 12-myristate 13-acetate (PMA;0, 10, 25, 50, 100, and 200 ng/mL), the classic activator of PKC, for 48 h. Compared with the cells with no PMA treatment, 50 and 100 ng of PMA/mL significantly stimulated the glucose uptake of the BMEC, whereas the glucose uptake by the cells treated with the lowest and the highest amounts of PMA (25 and 200 ng/mL, respectively) did not show a significant difference. Consistently, the mRNA expression of glucose transporter (GLUT) 1 and 8 showed a similar pattern of increase under the treatments of PMA. Furthermore, when the cells were pretreated with GF1090203X (0, 0.25, 0.5, 1, and 2 μM), an inhibitor of PKC, for 30 min before exposed to PMA (50 ng/mL), the PMA-induced glucose uptake and GLUT1 and GLUT8 expression were decreased by GF1090203X in a dose-dependent manner. These results demonstrate that PKC is involved in the regulation of glucose uptake by BMEC, and this function may work, at least partly, through upregulating the expression of GLUT1 and GLUT8.     

造纸法再造烟叶加工过程中5种甾醇的含量变化

为研究造纸法再造烟叶各工艺阶段甾醇类化合物的含量变化,利用超高效液相色谱-串联质谱仪(UPLC-MS/MS)对5种甾醇(麦角甾醇、胆甾醇、菜油甾醇、豆甾醇及β-谷甾醇)进行了测定。结果表明:①烟叶原料中5种甾醇的含量(质量分数)均高于烟梗,且两种烟草原料中的甾醇类化合物均以豆甾醇和β-谷甾醇为主;②在提取和浓缩阶段,甾醇含量均降低,降低量分别占原料中甾醇总量的21.35%和15.13%;③在掺配阶段,低浓浆中甾醇实际增加量占原料中甾醇总量的10.64%;④在打浆和抄造阶段,甾醇含量均降低,降低量分别占原料中甾醇总量的14.06%和14.18%;⑤其余工艺阶段的甾醇含量变化不大,最终再造烟叶产品中甾醇量占原料中甾醇总量的48.67%。      

In vitro antioxidant and anti-inflammatory effects of brewers' spent grain protein rich isolate and its associated hydrolysates

Brewers' spent grain (BSG) is a protein-rich by-product of the brewing industry. The present study examined the in vitro bioactivity of a BSG protein enriched preparation and its associated enzymatic hydrolysates (assigned A–J). Cytotoxicity was measured using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay in U937 and Jurkat T cells. IC₅₀ values were lower in the U937 cell line, ranging from 4.93 to 9.27% v/v versus a range of 4.11% v/v to undetectable in Jurkat T cells. The superoxide dismutase (SOD) and comet assays were performed on U937 cells pre-incubated with test samples and subsequently exposed to an oxidant. Hydrogen peroxide (H₂O₂) significantly reduced SOD activity by 37.7% and none of the test samples provided protection. None of the samples protected against DNA damage induced by tert-butylhydroperoxide (t-BOOH); hydrolysate H, prepared with Alcalase at 60 °C, protected against H₂O₂-induced DNA damage. The total phenolic content (TPC) was found to range from 0.021 to 0.055 mg GAE/mg dry powder. The effect of the BSG-derived test samples on cytokine production (IL-2, IL-4, IL-10, IFN-γ) in Concanavalin A (conA) stimulated Jurkat T cells was measured using an enzyme linked immunosorbent assay (ELISA). The samples had no effect on IL-2 and IL-4 production. The unhydrolysed sample C significantly reduced IL-10, while the protein rich isolate, unhydrolysed control samples and hydrolysates D, E, F, and J significantly reduced IFN-γ production. The BSG preparations possess little antioxidant potential and exhibit selective immunomodulatory effects that may be of benefit in the control of inflammatory diseases.     

Analysis of free and esterified sterols in edible oils by online reversed phase liquid chromatography-gas chromatography (RPLC-GC) using the through oven transfer adsorption desorption (TOTAD) interface

An online reversed phase liquid chromatography-gas chromatography (RPLC-GC) method is proposed to quantify free, total and esterified sterols of edible oils. To determine free sterols the diluted oils are injected into the liquid chromatograph, where free sterols are separated from triglycerides and the sterol fraction is automatically transferred to the gas chromatograph to be analysed. To determine total sterols the samples were saponified with potassium hydroxide in ethanolic solution and the unsaponifiable fraction was extracted with diethyl ether. The extract was then analysed by RPLC-GC, avoiding the laborious thin layer chromatography step used in the Official European Union (EU) Method. The relative standard deviations (RSDs) from the absolute peak area varied from 7.6% to 15.8%. Limits of detection (LODs) were less than 8.5 mg/kg. No variability in retention time was observed. The method was applied to the determination of total sterols in edible oil samples and the results were compared with those obtained with the Official EU Method. Good agreement was found between both methods, except in the case of campesterol.     

Assessment of the reproductive-endocrine disrupting potential of chlorine dioxide oxidation products of plant sterols

This study examined the hypothesis that chlorine dioxide bleaching used in pulp and paper production causes the formation of reproductive-endocrine disrupting compounds from plant sterols. This was tested by conducting a laboratory simulation of the chlorine dioxide oxidation of two plant sterols, beta-sitosterol and stigmasterol. Oxidation products of the plant sterol beta-sitosterol were purified and identified and found to be cholestan-24-ethyl-3-one, 4-cholestene-24-ethyl-3-one, and 4-cholestene-24-ethyl-3,6-dione. The first two compounds were found in a number of pulp and paper effluents and biosolids. The sterols and their oxidation products were tested in vitro using bioassays for androgenicity and estrogenicity. A 28 d in vivo bioassay was employed to examine masculinization in female mosquitofish. In vitro bioassays revealed little estrogenic activity in the parent sterols or in mixtures of their oxidation products. Androgenic activity as measured by the androgen receptor binding bioassay was in the order of 19-96 microg/g testosterone equivalents but with no increase or decrease with chlorine dioxide oxidation. The mosquitofish bioassay did not show significant masculinization for any of the preparations tested. A number of androstane steroids were identified in the sterols tested, however, those compounds could only account for a small fraction of the androgenic activity in the sterols. It was clear that the parent sterols were not themselves acting as androgens in the bioassays used. This study indicated that chlorine dioxide oxidation of sterols produced predominantly oxidized sterols that were not likely to act through androgenic or estrogenic mechanisms.     

Sterols of seed oils of Vernonia galamensis,Amaranthus cruentus,Amaranthus caudatus,Amaranthus hybridus and Amaranthus hypochondriacus grown in the humid tropics

Analysis of sterols of seed oils from Vernonia galamensis, Amaranthus cruentus, A caudatus, A hybridus and A hypochondriacus, the last four being exotic breeds planted in the humid tropics of Africa, is presented in this report. Identifiable sterols in all the seed oil samples include campesterol, stigmasterol, ß-sitosterol, Δ⁵-avenasterol and Δ⁷-avenasterol except for Vernonia galamensis where cholesterol was detected (63.61 mg per 100 g oil).     

Expression of urokinase plasminogen activator receptor in resting and activated bovine neutrophils

Changes in urokinase-plasminogen activator (u-PA) and u-PA receptor (u-PAR) expression at the protein and mRNA level in resting neutrophils and in neutrophils activated by phorbol myristate acetate (PMA) were examined. Low amounts of u-PA were found intracellularly or membrane-bound in resting neutrophils. However, incubation of resting neutrophils with purified exogenous u-PA (10 IU/ml) revealed extensive binding of u-PA to cell membranes. Excess amino-terminal fragment of the u-PA molecule, a proteolytically inactive fragment of u-PA (amino acids 1-135) blocked binding of exogenous u-PA to the cell membrane. These results, collectively, indicate that the binding of u-PA is specific and that resting neutrophils have unoccupied u-PA receptors on their cell membrane. Addition of PMA led to an increase (P < 0.01) in total cell-associated, membrane-bound u-PA activity and u-PA mRNA expression by bovine neutrophils. In contrast. PMA increased u-PAR mRNA levels but this was accompanied by a decrease (2.5-fold; P < 0.01) in free, unoccupied u-PA binding sites. No significant effects on total cell-associated or membrane-bound u-PA were found when neutrophils were treated with 4-phorbol 12,13 didecanoate, a phorbol ester that does not activate protein kinase C (PKC). Furthermore, addition of 1-(5-isoquinolinesylphonyl)-2-methlylpiperazine dihydrochloride (H-7), a potent PKC inhibitor, blocked the effect of PMA on total cell-associated u-PA activity. Thus, PKC plays a role in the modulation of u-PA and u-PAR by PMA in bovine neutrophils.