In Situ Preparation of Fatty Acid Methyl Esters for Analysis of Fatty Acid Composition in Foods

On the basis of the isomer-specific effects of trans fatty acids (FA) on human health, and the detrimental effect of t10,c12-conjugated linoleic acid (CLA) on cows' milk fat production, there is a need to identify factors that affect the shift from trans-11 to trans-10 pathway during ruminal biohydrogenation of FA. This experiment was conducted in vitro and aimed at separating the effects of the diet of the donor cows from those of the fermentative substrate, which is necessary to prevent this shift. A total of four dry Holstein dairy cows were used in a 4 3 4 Latin square design. They received 12 kg of dry matter per day of four diets based on maize silage during four successive periods: the control diet (22% starch, ,3% fat); the high-starch diet, supplemented with wheat plus barley (35% starch, ,3% crude fat); the sunflower oil diet, supplemented with 5% of sunflower oil (20% starch, 7.6% crude fat); and the high-starch plus oil diet (33% starch, 7.3% crude fat). Ruminal fluid of each donor cow was incubated for 5 h with four substrates having similar chemical composition to the diets, replacing sunflower oil by pure linoleic acid (LA). The efficiency of isomerisation of LA to CLA was the highest when rumen fluids from cows receiving dietary oil were incubated with added LA. The shift from trans-11 to trans-10 isomers was induced in vitro by high-starch diets and the addition of LA. Oil supplementation to the diet of the donor cows increased this shift. Conversely, the trans-10 isomer balance was always low when no LA was added to incubation cultures. These results showed that a large accumulation of trans-10 FA was only observed with an adapted microflora, as well as an addition of non-esterified LA to the incubation substrate.

Section: Methodsmentioning

confidence: 99%

In vitro study of dietary factors affecting the biohydrogenation shift from trans-11 to trans-10 fatty acids in the rumen of dairy cows

Zened

Enjalbert

Nicot

et al. 2012

“…Total lipids were extracted from a pellet of adipocytes as well as 1 ml of spent media using chloroform : methanol (2:1 v/v) as described by Folch et al (1957). Methylation was carried out for both adipocytes and spent media using the method described by Park and Goins (1994). The FAME were separated on a CP Sil 88 column (100 m 3 0.25 mm i.d., 0.20 mm film thickness; Chrompack, Middleburg, The Netherlands) using a gas liquid chromatograph (3400; Varian, Harbor City, CA, USA) fitted with a flame ionization detector.…”

Section: Methodsmentioning

confidence: 99%

Effect of level of eicosapentaenoic acid on the transcriptional regulation of Δ-9 desaturase using a novel in vitro bovine intramuscular adipocyte cell culture model

Waters

Kenny²,

Killeen³

et al. 2009

Ruminant fat is often perceived as having a negative impact on human health; however, the composition of the fat is under complex biochemical control and can be improved through strategic manipulation of the animal's diet. There were two major objectives of this study, namely (i) to develop and validate a primary bovine intramuscular adipocyte cell line and (ii) to examine the effect of eicosapentaenoic acid (EPA) on the transcriptional regulation of D-9 desaturase in vitro using the novel cell line. Intramuscular adipose tissue was obtained from the Musculus longissimus thoracis of a beef heifer. Mature adipocytes were isolated and cultured, and subsequently harvested and evaluated for lipid accumulation and the expression of genes regulating key functional adipocyte protein markers at passages 10, 20 and 30. Isolated cells were shown to accumulate lipid in culture over time. Fatty acid analysis by gas chromatography was carried out at passage 30. Thirteen fatty acids ranging from tetradecanoic acid (C14:0) to the polyunsaturated fatty acid, docosahexaenoic acid (C22:6), were easily detected and measured. High-quality total RNA was isolated from adipocytes and the expression of peroxisome proliferator-activated receptor-g, fatty acid synthase, fatty acid-binding protein-4, adipocyte lipid-binding protein, CD36, D-9 desaturase, sterol regulatory element-binding protein (SREBP), microsomal triglyceride transfer protein and leptin genes were identified by reverse transcriptase-PCR and sequence analysis. Expression of the negative control, liver-specific hepatocyte nuclear factor-1alpha, was not detected. Adipocytes were subsequently incubated in medium containing 0, 50 or 100 mM EPA for 24 h. Increasing the EPA concentration of the culture media led to a linear increase in adipocyte EPA concentration ( P , 0.01). Expression of D-9 desaturase mRNA was decreased five-and seven-fold, respectively, following 50 and 100 mM EPA incubation compared to the control. Gene expression of SREBP-1c was decreased by 6-and 18-fold in cells supplemented with 50 and 100 mM EPA, respectively, compared to the control. Regression analysis showed a negative linear relationship between EPA concentration and the gene expression of both D-9 desaturase ( P , 0.001) and SREBP-1c ( P , 0.001), while a significant positive relationship was observed between D-9 desaturase and SREBP-1c gene expression ( P , 0.001). This is the first report demonstrating that EPA treatment of bovine intramuscular adipocyte cells decreased gene expression of both D-9 desaturase and SREBP-1c in vitro. The bovine adipocyte cell line developed here is an important resource for future studies facilitating less-expensive, rapid screening of research hypotheses and circumventing the limitations associated with the use of experimental animals including cost, inter-animal variation, pre-experimental management and ethics.

“…Plasma total lipids were extracted as described by Folch et al (1957) and 19:0 was used as an internal standard. FAs in plasma lipids extracts and in unextracted diet ingredients and milk samples were methylated using sodium methoxide followed by boron trifluoride as described by Park and Goins (1994). This method, which successively uses basic and acid transmethylations, allows methylation of all lipid classes, including non-esterified fatty acids, and does not alter the stereochemistry of CLA double-bonds (Duckett et al, 2002 For plasma analysis, flame ionisation detector temperature was maintained at 2608C and the injector at 2558C, and a splitless injection with an automatic injector was used.…”

Section: Methodsmentioning

confidence: 99%

Conjugated linolenic acid (CLnA), conjugated linoleic acid (CLA) and other biohydrogenation intermediates in plasma and milk fat of cows fed raw or extruded linseed

Akraim

Nicot

Juanéda

et al. 2007

Thirty lactating dairy cows were used in a 3 3 3 Latin-square design to investigate the effects of a raw or extruded blend of linseed and wheat bran (70:30) on plasma and milk fatty-acids (FA). Linseed diets, containing 16.6% linseed blend on a dry-matter basis, decreased milk yield and protein percentage. They decreased the proportions of FA with less than 18 carbons in plasma and milk and resulted in cis-9, cis-12, cis-15 18:3 proportions that were more than three and four times higher in plasma and milk, respectively, whereas cis-9, cis-12 18:2 proportions were decreased by 10-15%. The cis-9, trans-11, cis-15 18:3 isomer of conjugated linolenic acid was not detected in the milk of control cows, but was over 0.15% of total FA in the milk fat of linseed-supplemented cows. Similarly, linseed increased plasma and milk proportions of all biohydrogenation (BH) intermediates in plasma and milk, including the main isomer of conjugated linoleic acid cis-9, trans-11 18:2, except trans-4 18:1 and cis-11, trans-15 18:2 in plasma lipids. In milk fat, compared with raw linseed, extruded linseed further reduced 6:0-16:0 even-chain FA, did not significantly affect the proportions of 18:0, cis-9 18:1 and cis-9, cis-12 18:2, tended to increase cis-9, cis-12, cis-15 18:3, and resulted in an additional increase in the proportions of most BH intermediates. It was concluded that linseed addition can improve the proportion of conjugated linoleic and linolenic acids, and that extrusion further increases the proportions of intermediates of ruminal BH in milk fat.