Mertxe de Renobales

DNA methylation epigenotypes in breast cancer molecular subtypes

IntroductionIdentification of gene expression-based breast cancer subtypes is considered a critical means of prognostication. Genetic mutations along with epigenetic alterations contribute to gene-expression changes occurring in breast cancer. So far, these epigenetic contributions to sporadic breast cancer subtypes have not been well characterized, and only a limited understanding exists of the epigenetic mechanisms affected in those particular breast cancer subtypes. The present study was undertaken to dissect the breast cancer methylome and to deliver specific epigenotypes associated with particular breast cancer subtypes.MethodsBy using a microarray approach, we analyzed DNA methylation in regulatory regions of 806 cancer-related genes in 28 breast cancer paired samples. We subsequently performed substantial technical and biologic validation by pyrosequencing, investigating the top qualifying 19 CpG regions in independent cohorts encompassing 47 basal-like, 44 ERBB2+ overexpressing, 48 luminal A, and 48 luminal B paired breast cancer/adjacent tissues. With the all-subset selection method, we identified the most subtype-predictive methylation profiles in multivariable logistic regression analysis.ResultsThe approach efficiently recognized 15 individual CpG loci differentially methylated in breast cancer tumor subtypes. We further identified novel subtype-specific epigenotypes that clearly demonstrate the differences in the methylation profiles of basal-like and human epidermal growth factor 2 (HER2)-overexpressing tumors.ConclusionsOur results provide evidence that well-defined DNA methylation profiles enable breast cancer subtype prediction and support the utilization of this biomarker for prognostication and therapeutic stratification of patients with breast cancer.

Immobilization of Candida rugosa lipase and some properties of the immobilized enzyme

Lipase (triacylglycerol ester hydrolase, E.C.3.1.1.3) from Candida rugosa has been immobilized on commercially available microporous polypropylene. The enzyme was rapidly adsorbed on the support, and more than 60% of the soluble activity disappeared from the medium after 1 min of incubation at room temperature. A recovery of immobilized activity of 21% was obtained when the wet preparation was immediately assayed with olive oil at the end of the immobilization protocol. The activity of the immobilized enzyme drastically decreased with the loss of water of the preparation. Pretreatment of the support with organic solvents significantly increased the recovered immobilized activity. Our results strongly suggest that the soluble lipase could exist in different aggregation forms depending on the pH of the medium. At acidic pH, the relative proportion of high-molecular-weight forms of the enzyme is higher than at pH 7.0, suggesting that the lipase would be also immobilized in different aggregation forms depending on the pH used in the immobilization procedure. Crosslinking of the adsorbed enzyme with glutaraldehyde diminished its activity but increased the stability of the lipase against the washing-out effect of Triton X-100. Data on the most relevant catalytic properties of the soluble and immobilized enzyme, such as optimum pH and temperature as well as ranges of stability, kinetic parameters, and activation energy for the hydrolysis of olive oil and p-nitrophenyl acetate, are reported.

Coagulating and lipolytic activities of artisanal lamb rennet pastes

Lamb rennet pastes were prepared by the procedure most commonly used by Idiazabal cheese manufacturers. We studied the effects on their coagulating and lipolytic activities of the state of the stomach at the time of death (full of milk or empty), the amount of NaCl added, the origin of the lambs and paste storage time. Coagulating activities were generally between 155 and 363 units/g tissue. Pastes prepared from stomachs of lambs from slaughterhouse flocks had significantly higher coagulating activities than those of lambs from separate flocks. No significant decrease in coagulating activity was observed after 1 year storage at 4 degrees C. Chymosin represented 75-80% of the total coagulating activity with the remainder being pepsin. Rennet paste extracts with pH < 4.7 did not have increased coagulating activities when their pH was lowered to 2.0, while those with pH > 5.2 had activities 1.5-fold those before treatment. Lipase activity was higher in extracts of rennet pastes prepared using the stomachs of lambs that arrived at the slaughterhouse in the morning just prior to slaughter than in those prepared with the stomachs of lambs that had arrived on the previous evening. However, the reverse was the case for esterase activity. Activating the coagulating activity by pH cycling completely destroyed both lipolytic activities. Storage at 4 degrees C for > 1 year did not affect esterase activity but lipase activity decreased substantially after 4-5 months. Lipase, but not esterase, activity was responsible for the liberation of short-chain free fatty acids from ovine milk fat.

Determination of free fatty acids in cheese: comparison of two analytical methods

Two methods were compared for the determination of free fatty acids (FFA) from acetic to long-chain acids in samples with a large excess of triacylglycerols (TG) (1[ratio ]200, w/w), such as cheese and other dairy products. In method 1, after fat extraction, FFA were separated from TG by aminopropyl-bonded phase chromatography, injecting the fraction containing FFA directly into the gas chromatograph. In method 2, extracted fat was treated with tetramethylammonium hydroxide, the methyl ester derivatives being formed in the injector. Cheese samples and standard mixtures of FFA and TG in different proportions were analysed by both methods. The cheese sample contained 2·4 times more FFA when analysed by method 2 as compared with the result obtained with method 1. The composition of the standard mixtures analysed by method 1 closely reflected that of the original mixture and gave 90–100% recovery of FFA, regardless of their chain length and the ratio of FFA[ratio ]TG (1[ratio ]1 or 1[ratio ]200, w/w). The composition of samples with a FFA[ratio ]TG ratio of 1[ratio ]200 (w/v) was severely distorted (as compared with the original composition of the sample) when analysed by method 2. Varying recoveries of FFA were also obtained, the largest differences being found for the shorter-chain components. We conclude that the FFA fraction should be separated from the TG fraction before derivatization and chromatographic analysis, particularly for samples in which the FFA represent a minor fraction of the TG.

Hydrolysis of animal fats by immobilized Candida rugosa lipase

Lipase (triacylglycerol ester hydrolase, EC 3.1.1.3) from Candida rugosa was immobilized by adsorption on a commercially available microporous polypropylene support of 200- to 400-μm particle size. A contact period of 90 min allowed the highest degrees of hydrolysis to be achieved, particularly in the second and third hydrolysis reactions. The optimal hydrolysis conditions were 0.10 kg enzyme per kilogram fat, 50% (w/v) fat, and 40°C for 24 h. The immobilized enzyme can be repeatedly used and hydrolysis degrees of 90% or higher can be achieved. Of the three animal fats studied, edible pork lard consistently yielded the highest degrees of hydrolysis (95%) in the first hydrolysis reaction and inedible beef tallow the lowest (65%). The immobilized enzyme lost its activity above 45°C. The support could be easily recovered and reused up to 5 times.

Seasonal changes in the composition of bulk raw ewe's milk used for Idiazabal cheese manufacture

Seasonal changes in the composition of bulk raw ewes milk used for Idiazabal cheese manufacture and its influence on the cheese yield are reviewed. This review is based on biochemical and microbiological data previously published on the composition of a bulk raw ewes milk collected at three different times of the year. In order to characterize, in a more extensive perspective, the seasonal changes in the composition of this bulk raw ewes milk and its influence on actual cheese yield, multivariate statistical analyses were applied to the compositional data of the milk and the actual cheese yield obtained at the factory. The results show pronounced seasonal changes in the composition of bulk raw ewes milk. Changes in cheese yield, lipolytic activity and microbiological quality of the bulk raw ewes milk are the principal aspects seasonally affected during the cheesemaking period. Also, the seasonal changes observed in the bulk raw ewes milk composition strongly affect the quality of Idiazabal cheeses manufactured over the cheesemaking period.

Habitual fish intake is associated with decreased LDL susceptibility to ex vivo oxidation

A sample of 101 free-living individuals eating their habitual diets had fish consumptions ranging from less than one serving per week to over five servings per week. Statistically significant positive correlations were found between the amounts of EPA (20:5), DHA (22:6), and total n-3 PUFA ingested with the diet and their amounts in serum and in the phospholipid and cholesterol ester fractions of isolated LDL. No statistically significant correlations were observed between the intake and the serum or LDL amounts of any other FA [total n-6 PUFA, linoleic acid (18:2), arachidonic acid (20:4), monounsaturated FA, or saturated FA)]. The increase in serum n-3 PUFA did not affect the Trolox-equivalent antioxidant capacity of serum (1.18 ±0.17 mmol/l). When isolated LDI were subjected to Cu2+-induced ex vivo oxidation, a statistically significant but negative correlation was found between intake of n-3 PUFA and the rate of appearance of conjugated dienes as well as with the total amount of conjugated dienes. In contrast, intake of n-6 PUFA showed a significant and positive correlation with these two oxidation parameters. The observed results suggest that 22:6 but not 20:5 could have a possible protective effect, whereas perhaps 20:4 and 18:2 could have a prooxidant effect.

Enzymic hydrolysis of animal fats in organic solvents at temperatures below their melting points

Lipase fromCandida rugosa catalyzed the hydrolysis of inedible beef tallow and pork lard (edible and inedible) in the presence of organic solvents at temperatures below the melting point of the fat. Reactions were carried out at 50% substrate with 180 lipase units per gram of fat in a two-liter reactor. In the presence of isooctane (5-10%) beef tallow yielded 94% hydrolysis in 24 hr both at 37° and 31°C. Edible pork lard yielded 97% hydrolysis under these conditions and at temperatures as low as 25°C, while inedible lard gave hydrolysis intermediate between the other two fats.

Simultaneous analysis of carotenoids and tocopherols in botanical species using one step solid-liquid extraction followed by high performance liquid chromatography.

Carotenoids and tocopherols from botanical species abundant in Atlantic mountain grasslands were simultaneously extracted using one-step solid-liquid phase. A single n-hexane/2-propanol extract containing both types of compounds was injected twice under two different sets of HPLC conditions to separate the tocopherols by normal-phase chromatography and carotenoids by reverse-phase mode. The method allowed reproducible quantification in plant samples of very low amounts of α-, β-, γ- and δ-tocopherols (LOD from 0.0379 to 0.0720 μg g(-1) DM) and over 15 different xanthophylls and carotene isomers. The simplified one-step extraction without saponification significantly increased the recovery of tocopherols and carotenoids, thereby enabling the determination of α-tocopherol acetate in plant samples. The two different sets of chromatographic analysis provided near baseline separation of individual compounds without interference from other lipid compounds extracted from plants, and a very sensitive and accurate detection of tocopherols and carotenoids. The detection of minor individual components in botanical species from grasslands is nowadays of high interest in searching for biomarkers for foods derived from grazing animals.

Winter/Spring Changes in Fatty Acid Composition of Farmhouse Idiazabal Cheese Due to Different Flock Management Systems

Typically, two different flock managements are employed by basque sheepherders in winter and spring. Thus, seasonal changes in the fatty acid (FA) composition of Idiazabal PDO farmhouse cheeses were studied. Ewes raw milk cheeses elaborated in winter and spring were collected after 120 days of ripening from 10 Idiazabal PDO farmhouses. In winter, concentrate and conserved forages were fed, whereas a part-time grazing system was adopted from spring onward. Spring cheeses had less (P <or= 0.05) saturated FA and higher (P <or= 0.05) content of unsaturated FA, including trans-FA (mainly trans-vaccenic acid) and conjugated linoleic acid (CLA), branched-chain FA (BCFA), and n-3 FA. Principal component analysis (PCA) separated winter and spring cheeses into two groups by the combination of two principal components (84.2% of variance). Fresh pasture in the diet enhanced desirable FA and lowered atherogenicity index in cheeses, supporting the benefits of using a part-time grazing system for the consumer.