The Sum of Plasma Fatty Acids iso16:0, iso17:0, trans11-18:1, cis9, trans11-CLA, and cis6-18:1 as Biomarker of Dairy Intake Established in an Intervention Study and Validated in the EPIC Cohort of Gipuzkoa.

The questioned reliability of 15:0, 17:0, and trans9-16:1 acids as biomarkers of dairy fat intake also questions the relationship between the intake of these products and their health effects. Two studies were conducted in the same geographical region. In an intervention study, volunteers followed a diet rich in dairy products followed by a diet without dairy products. Plasma and erythrocyte fatty acids (FA) were analyzed, and their correlations with dairy product intakes were tested. The FA biomarkers selected were validated in the Gipuzkoa cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC) observational study. The correlation coefficients between plasma concentrations of iso16:0, iso17:0, trans11-18:1, cis9, trans11-18:2, and cis6-18:1 and the dairy fat ingested are similar in both studies, indicating that their concentration increases by 0.8 µmol/L per gram of dairy fat ingested. The biomarkers are positively related to plasma triglycerides (r = 0.324 and 0.204 in the intervention and observational studies, respectively) and total cholesterol (r = 0.459 and 0.382), but no correlation was found between the biomarkers and atherogenicity indexes. In conclusion, the sum of the plasma concentration of the selected FAs can be used as biomarkers of dairy product consumption. A linear relationship exists between their plasma concentrations and ruminant product intake. These biomarkers allow for obtaining consistent relationships between dairy intake and plasma biochemical parameters.

Changes in terpenoid composition of milk and cheese from commercial sheep flocks associated with seasonal feeding regimens throughout lactation.

Changes in the terpenoid content of milk and cheese from commercial sheep flocks monitored throughout lactation in the Cantabrian area of northern Spain were investigated. The flocks followed the same seasonal feeding strategy during lactation: indoor feeding in winter (early lactation) based on concentrate and forage; part-time grazing in the valley in early spring (mid lactation); and from mid spring on (late lactation), flocks were managed under extensive mountain grazing. In the present study design, seasonal feeding and lactation stage were intrinsically linked and could not be considered in isolation, and a holistic approach was necessary to consider the whole production management of the commercial flocks studied. Furthermore, the study focused on the identification of sesquiterpenoid ratios to differentiate milks and cheeses produced under extensive mountain grazing from those produced under other seasonal feeding regimens. Total abundance of mono- and sesquiterpenoids and that of individual compounds such as α-pinene, ß-caryophyllene, α-humulene, α-amorphene, and γ-cadinene significantly increased in milk and cheese from indoor feeding to mountain extensive grazing. Sesquiterpenoid ratios such as γ-cadinene/α-muurolene, γ-cadinene/δ-cadinene, ß-caryophyllene/α-muurolene, and (ß-caryophyllene + γ-cadinene)/α-muurolene were used to differentiate mountain milks and cheeses from those from indoor feeding and part-time grazing in the valley. Multivariate discriminant analysis applied to individual terpenoids and sesquiterpenoid ratios showed milk and cheese samples classified into 2 groups: samples from indoor feeding and part-time grazing in the valley were classified together, and clearly separated from mountain milks and cheeses. The results of the present study showed that the sesquiterpenoid ratios approach could help to differentiate mountain dairy products from others obtained under other specific feeding regimens in a local environment.

Commercial sheep flocks--fatty acid and fat-soluble antioxidant composition of milk and cheese related to changes in feeding management throughout lactation.

Fatty acids (FAs), tocopherols and retinoids were analysed in raw milk and cheese from six commercial sheep flocks monitored from early lactation in winter to late lactation in summer. In winter, animals received concentrate and forage indoors; in early spring, animals grazed part-time on cultivated or natural valley grasslands; and from mid spring on, animals were kept outdoors constantly on mountain natural pastures. Mountain grazing in late lactation significantly increased the amount of healthy desirable unsaturated FAs such as C18:1t11 (VA), C18:2c9t11 (RA), C18:2t11c13, C18:3c9c12c15 (ALA) and C20:5c5c8c11c14c17 (EPA), and those of α-tocopherol and α-tocotrienol of milk and cheese. Stepwise discriminant analysis was applied to classify cheese samples according to seasonal feeding management. The multivariate approach was able to discriminate beyond doubt mountain cheeses from those of indoor feeding and part-time valley grazing.

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-, ß-, 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.

Rapeseed and sunflower oilcake as supplements for dairy sheep: animal performance and milk fatty acid concentrations.

The influence of different amounts of oilseed cake (rapeseed and sunflower) on animal production parameters and fatty acid (FA) concentrations of the milk was studied in a Latxa dairy sheep experimental flock, both in winter (50% oilcakes; indoor feeding) and in spring (30% oilcakes; part-time grazing). The two different levels of the oilcakes tested did not affect animal production parameters or milk yield. Milk fat content and the fat/protein ratio decreased significantly with 30 and 50% sunflower cake. Yet, fat/protein ratio values were within the range for cheesemaking. Both levels of either type of oilcake tested significantly increased the concentrations of nutritionally interesting FA (CLA isomer C18:2cis-9, trans-11, vaccenic, oleic, and total unsaturated FA), while simultaneously decreasing the concentration of atherogenic FA. The atherogenicity indexes of milks from ewes fed 50 or 30% of either oilcake were significantly lower than those of their corresponding control. The use of cakes in winter increased the concentration of nutritionally interesting FA to the values obtained with part-time grazing.

Interannual and geographical reproducibility of the nutritional quality of milk fat from commercial grazing flocks.

The objective of the present work was to study the differences in the fatty acid (FA) composition of raw sheep milk fat under commercial milk production conditions throughout lactation, in two consecutive years. Particular attention was placed on the C18:2cis-9,trans-11 isomer, C18:1trans-11 acid, and unsaturated FA as the feeding regimen of 10 commercial flocks of latxa dairy sheep changed from indoor feeding to part-time grazing conditions (from early spring) as traditionally practiced in the Basque Country (Northern Spain). Farms located at an altitude of between 600 and 700 m, in two different geographical areas with different rainfall were selected. Milk samples were collected monthly from late January (indoor feeding) until mid-, or end of, June (outdoor feeding), during two consecutive years. In spite of some interannual variability (most likely due to large differences in rainfall), the evolution of individual FA throughout lactation was comparable between years, indicating that it was reproducible under commercial milk production conditions. The average concentrations of C18:2cis-9,trans-11 isomer and C18:1trans-11 acid in milk from the commercial flocks increased about 200% during the transition period (end of March or early April until May), from indoor feeding (late January or early February until the end of March) to the outdoor period (early May to mid-June), remaining constant during the outdoor period (27·53 ± 9·32 µmol/g fat and 71·58 ± 20·53 µmol/g fat, respectively). Non-atherogenic FA comprised approximately 50% of all saturated FA at any time during lactation, whereas the milk atherogenicity index decreased significantly during the outdoor period. The Trolox-equivalent antioxidant capacity of the water-soluble milk fraction did not appear to be influenced by feeding management. The FA composition of cheeses made during the second year with milk from the indoor or outdoor periods reflected those of the corresponding milks. A principal components analysis clearly showed that differences in the milk FA composition were primarily due to outdoor grazing, with very little contribution from the geographical zone or the year.

Effects of seasonal changes in feeding management under part-time grazing on terpene concentrations of ewes' milk.

Terpene composition of ewes' raw milk from nine commercial flocks was analysed from February to July. Ewes' diet consisted of concentrate and conserved forage in winter (indoor feeding) and part-time grazing from spring (transition and outdoor feeding). Regardless of the feeding, limonene and ß-phellandrene were the most abundant monoterpenes and ß-caryophyllene showed the highest concentrations among sesquiterpenes. Terpene content increased in the milks of commercial flocks when animals were reared under grazing management. Monoterpenes were detected in the milks of all the commercial flocks throughout the season, whereas sesquiterpenes were only detected in the milks from flocks grazing on non-cultivated community-owned grasslands in which a higher biodiversity of plant species grew. These preliminary results indicated that ß-caryophyllene could be a potential pasture-diet marker in the case of milks from animals grazing a higher biodiversity of plant species but in-depth studies including information on terpene composition of plants ingested by the animals are necessary to evaluate the suitability of ß-caryophyllene or another terpenoid compound as pasture biomarker.

DNA methylation epigenotypes in breast cancer molecular subtypes.

INTRODUCTION: Identification of gene expression based breast cancer subtypes is considered as 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 there is only a limited understanding of the epigenetic mechanisms affected in those particular breast cancer subtypes. The present study was undertaken to dissect the breast cancer methylome and deliver specific epigenotypes associated with particular breast cancer subtypes. METHODS: 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 biological 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. Using all-subset selection method, we identified the most subtype predictive methylation profiles in multivariable logistic regression analysis. RESULTS: The approach efficiently recognized 15 individual CpG loci differentially methylated in breast cancer tumor subtypes. We further identify novel subtype specific epigenotypes which clearly demonstrate the differences in the methylation profiles of basal-like and human epidermal growth factor 2 (HER2)-overexpressing tumors. CONCLUSIONS: Our results provide evidence that well defined DNA methylation profiles enables breast cancer subtype prediction and support the utilization of this biomarker for prognostication and therapeutic stratification of patients with breast cancer.

Seasonal changes in the technological and compositional quality of ewe's raw milks from commercial flocks under part-time grazing.

Rennet coagulation parameters, curd texture and gross compositional variables were studied in ewes' raw milk samples from nine commercial flocks using different concentrate:forage ratios and grazing times. From early lactation to March flocks were fed concentrate pellets and hay whereas from April to the end of lactation flocks were allowed to graze from 6 to 19 h/day receiving concentrate supplementation in the morning and evening. Milk from late-lactation flocks, when allowed to graze, showed higher content of fat, dry matter, protein, casein, soluble protein, total calcium, curd firmness and curd resistance to compression than the milk from early lactation flocks. Higher total calcium content and lower fat content were found when the early lactation flocks were fed high concentrate:forage ratio than when the flocks were fed low ratio. Curd firmness were lower for milk from flocks fed high concentrate:forage ratio, and the curd resistance to compression was greater, than for milk from flocks fed low ratio. At late lactation, when flocks grazed for a long time per day the total calcium content was higher than when the flocks grazed for a short time per day. Principal component analysis showed that protein and fat content were highly correlated with coagulum and curd firmness, whereas total calcium content was highly correlated with curd resistance to compression, and milk pH with rennet coagulation time.

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. Ewe's 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

GSTT1 and GSTM1 gene copy number analysis in paraffin-embedded tissue using quantitative real-time PCR.

GSTT1 and GSTM1 genes possess an inherited deletion associated with a lack of enzyme activity. The heterozygous condition of this deletion is difficult to determine in low-quality DNA with existing PCR protocols. We designed and validated a multiplex real-time PCR assay by adapting the DeltaDeltaCt relative quantification method for the analysis of GSTT1 and GSTM1 markers to accurately differentiate the three genotypes ( *1/1, *1/0, and *0/0) in degraded DNA from formalin-fixed paraffin-embedded tissue. Gene copy number values obtained provide for unambiguous homozygous and heterozygous differentiation. The efficacy shown by the PCR assay endorses its usefulness for complete genotyping of glutathione S-transferases in archival tissues.

Hygienic quality of ewes' milk cheeses manufactured with artisan-produced lamb rennet pastes.

The use of artisan-produced lamb rennet pastes, but not any of the other commercial animal rennets, imparts a characteristic flavour to the cheese, so most Mediterranean ewes' milk cheeses are coagulated with this kind of rennet paste. In contrast to the advantages of using lamb or kid rennet pastes from the sensory point of view, questions are still raised as to their hygienic quality. The goal was to examine the microbiological and enzymic quality of lamb rennet pastes prepared by cheese manufacturers for their own use, and evaluate the hygienic quality of raw sheeps' milk cheeses made with them, using Idiazabal cheese as a model. Lamb rennet pastes prepared by artisan cheese makers from the Basque region of Spain (27), and Italy (8) were evaluated. For cheese making experiments 5 different lamb rennet pastes were selected among the 27 samples from the Basque Country region of Spain. Microbiological analyses were carried out on samples from rennet pastes, rennet extracts, milks and cheeses during ripening. Enzymic activities studied in rennet paste were: total coagulating strength and lipase. Analysis of variance and Student's t-tests was performed. The results show that the artisan-produced rennet pastes contain high levels of a variety of microorganisms. After 60 ripening days, which is the minimum ripening period required for Idiazabal cheese prior to its commercialization, no Eschericia coli, Clostridium, Salmonella spp. or Listeria monocytogenes were detected, and levels for the rest of the microorganisms were below the limits of the European legislative standards for cheese manufactured with raw milk. We can conclude that the use of artisan-produced lamb rennet pastes of questionable hygienic quality for the manufacture of raw milk hard cheeses yields products of good hygienic quality.

Changes in lipid fractions and sensory properties of Idiazabal cheese induced by lipase addition.

This work studied the addition of an adequate lipase to enhance lipolysis reactions and the development of piquant flavour and sharp odour in Idiazabal cheese, as an alternative to the use of lamb rennet paste. Cheeses were manufactured from bulk raw ewes' milk in 50 l vats with commercial bovine rennet and 80 lipase units of pregastric or 180 lipase units of fungal lipase and ripened for 180 days. A higher lipolytic activity was induced by lipase addition promoting strong changes in odour and flavour attributes. Both fungal and pregastric lipases increased the content of total free fatty acids (FFA), but the fungal lipase released mainly medium- and long-chain FFA. In contrast, the pregastric lipase preferably released short-chain FFA. Diglyceride (DG) content was considerably higher in cheeses made with added pregastric lipase compared with those made with fungal lipase or with no lipase. Monoglycerides (MG) were detected only in cheeses made with either lipase added, reaching comparable concentrations after ripening for 180 days. The cheeses made with pregastric lipase had the highest scores for odour and flavour intensity, and sharp and rennet odours, desirable attributes for the Idiazabal cheese made with lamb rennet paste. None of the texture attributes were significantly influenced by the concentrations of MG and DG in the cheeses made with either lipase. Thus, the pregastric lipase was more appropriate than the fungal lipase to develop a more traditionally-flavoured Idiazabal cheese.