Changes in milk fat in response to dietary supplementation with calcium salts of trans-18:1 or conjugated linoleic fatty acids in lactating dairy cows.

Milk fat was investigated in lactating dairy cows fed diets supplemented with Ca salts of trans fatty acids (Ca-tFA) or Ca salts of conjugated linoleic acids (Ca-CLA). Forty-five Holstein cows (115 days in milk) were fed a control diet (51% forage; dry matter basis) supplemented with 400 g of EnerG II (Ca salts of palm oil fatty acids) for 2 wk; subsequently, 5 groups of 9 cows each were assigned for 4 wk to the control diet or diets containing 100 g of Ca-CLA or 100, 200, or 400 g of Ca-tFA in a randomized block design. Treatments had no effect on dry matter intake, milk production, protein, lactose, or somatic cell count. Milk fat percentage was reduced from 3.39% in controls to 3.30, 3.04, and 2.98%, respectively, by the Ca-tFA diets and to 2.54% by the Ca-CLA diet. Milk fat yield (1.24 kg/d in controls) was decreased by 60, 130, and 190 g/d with increasing dose of Ca-tFA and by 290 g/d with the Ca-CLA supplement. Consistent with increased endogenous synthesis of cis-9-containing CLA from precursors provided by the Ca-tFA diets, total CLA were similar in milk of cows fed Ca-CLA or Ca-tFA. Compared with controls, the Ca-CLA diet increased trans-10, cis-12-18:2 yield in milk, without altering levels of trans-18:1 isomers. In contrast, yields of most trans-18:1 isomers were elevated in milk of cows fed Ca-tFA diets, whereas yields of trans-10, cis-12-18:2 remained similar to control values. We conclude that milk fat depression can occur without an increase in trans-10, cis-12-18:2 in milk and that other components, perhaps the trans-10-18:1 isomer, may be involved.

Mammary lipogenic enzyme activity, trans fatty acids and conjugated linoleic acids are altered in lactating dairy cows fed a milk fat-depressing diet.

The objectives of the present study were to examine the effect of a milk fat-depressing (MFD) diet on: 1) the activity of mammary acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), 2) ACC mRNA relative abundance and 3) distributions of conjugated linoleic acids (CLA) and trans-18:1 fatty acids (tFA) in milk fat. Twelve lactating Holstein cows were used in a single reversal design. Two diets were fed: a control diet (60:40% forage/concentrate) and an MFD diet (25:70% forage/concentrate, supplemented with 5% soybean oil). The MFD diet decreased (P: < 0 0.001) milk fat by 43% and ACC and FAS activity by 61 and 44%, respectively. A reduced ACC mRNA relative abundance (P: < 0.001) corresponded with the lower ACC activity. The fatty acids synthesized de novo were decreased (P: < 0. 002), whereas tFA were increased from 1.9 to 15.6% due predominantly to a change in trans-10-18:1 isomer (P: < 0.001). With the MFD diet, the trans-7, cis-9 and trans-10, cis-12 CLA isomers were elevated (P: < 0.001), in contrast to the decrease in trans-11-18:1 (P: < 0. 001) and cis-9, trans-11-18:2. The data were consistent with a dietary effect on mammary de novo FA synthesis mediated through a reduction in ACC and FAS activity and in ACC mRNA abundance. The results were compatible with a role of trans-10, cis-12 CLA in milk fat depression, but alterations noted in tFA and other CLA isomers suggest that they also may be important during diet-induced milk fat depression.

Prenatal and postnatal transfer of fatty acids from mother to pup in the hooded seal.

Unlike most mammals, hooded seal (Cystophora cristata) pups are born with a substantial layer of adipose tissue. Subsequently, during the brief lactation period of only 4 days, fasting mothers mobilize enormous amounts of lipid from blubber and secrete milk (60% fat) at rates of 10 kg.day-1. Pups gain 7 kg.day-1 due primarily to the deposition of fat in blubber. We measured blubber content and fatty acid composition of blubber and milk in hooded seal mother-pup pairs at birth and over the 4-day lactation period to examine the nature and source of fetal lipids, the incorporation of maternal blubber fatty acids into milk lipid, and patterns of fatty acid deposition in suckling young. The fatty acid composition of the blubber of the newborn was notably different from that of its mother. Fetal deposition was likely due to a combination of both fetal synthesis and direct placental transfer of maternal circulating fatty acids. The blubber of the newborn was characterized by high levels (> 90% of total fatty acids) of saturated and monounsaturated fatty acids of primarily endogenous origin. In particular, the fetus appeared to have high delta-9 desaturase activity as evidenced by the large amounts of 14:1n-5 (4.2%) and 16:1n-7 (37.0%) in newborn blubber compared to maternal blubber (0.2% and 14.1%, respectively). Nevertheless, essential and long-chain polyunsaturated fatty acids of the n-3 and n-6 families, which could only have originated by direct transfer from the mother, comprised > 7% of pup blubber fatty acids and indicated greater rates of placental transfer than found in humans. In hooded seal mothers, rapid lipid transfer during the brief lactation period appeared to be facilitated by direct incorporation of mobilized fatty acids into milk. Although some differences in proportions of specific fatty acids were found between milk and maternal blubber, most of these differences declined over the course of lactation. However, selective mobilization of 20:5n-3 from maternal blubber into milk was apparent throughout lactation and resulted in elevated levels in pup blubber at weaning compared to maternal blubber. Ingested fatty acids were deposited directly and without modification into the blubber of pups, and by 4 days the fatty acid composition of pup blubber was virtually identical to that of the milk consumed.

Comparison of body weight and adipose tissue in male C57Bl/6J mice fed diets with and without trans fatty acids.

The effect of a diet containing trans-fatty acids (tFA) on the fatty acid composition and fat accumulation in adipose tissue was investigated in mice. Male C57Bl/6J mice were fed Control or Trans Diets that were similar, except that 50% of the 18:1, which was all cis in the Control Diet, was replaced by tFA in the Trans Diet. At selected ages, body weight, epididymal fat pad weight, perirenal fat yield, adipose tissue cellularity and fatty acid composition were examined. Over the time period studied (2-24 mon), the proportion of 18:0 and 16:0 tended to decrease while cis-18:1 levels increased. Compared to the Control Diet, the Trans Diet resulted in adipose tissue lipids with higher percentages of 14:0 and 18:2n-6 and lower percentages of cis-18:1 and 20:4n-6. In polar lipids, tFA replaced saturated fatty acids, whereas tFA replaced cis-18:1 in the nonpolar lipids. Body weights at 16 and 24 mon of age and epididymal fat pad weights at 8-24 mon of age were lower in mice fed the Trans Diet as compared to those fed the Control Diet. At the ages studied, the Trans Diet also resulted in lower values for perirenal fat weights, triacylglycerol to polar lipid ratios, and adipose cell size. The data suggest that chronic consumption of tFA affects lipid metabolism and results in decreased fat accumulation in murine adipose tissue.

Milk fat yield and composition during abomasal infusion of cis or trans octadecenoates in Holstein cows.

The role of trans-C18:1 fatty acids in milk fat depression was examined. Six rumen-cannulated Holstein cows were assigned to two Latin squares with 21-d periods. The common basal diet contained 40% forage and 60% concentrate. Treatments were the uninfused control, 750 g/d of a mixture of cis fat (65% high oleic sunflower oil and 35% cocoa butter), and 750 g/d of a mixture of trans fat (93% shortening and 7% corn oil) infused into the abomasum via a tube that passed through the rumen cannula. Milk yield was similar among treatments. Milk fat percentage and yield were lower, and milk citrate concentration was higher, for the trans than the cis treatment. Changes in the fatty acid composition of milk were similar for the cis and trans treatments compared with the control except for trans-C18:1. The concentration of trans-C18:1 was greater for the cis and trans treatments than for the control and was greater for the trans than for the cis treatment. These data clearly demonstrated that infusion of trans-C18:1 fatty acids into the abomasum depressed milk fat percentage and yield. We speculate that reduced synthesis of fatty acids and reduced activity of acyl transferase in mammary tissue contributed to depressed milk fat percentage for the trans treatment.

Comparison of body weight and adipose tissue in male C57BI/6J mice fed diets with and withouttrans fatty acids.

The effect of a diet containingtrans-fatty acids (tFA) on the fatty acid composition and fat accumulation in adipose tissue was investigated in mice. Male C57BI/6J mice were fed Control or Trans Diets that were similar, except that 50% of the 18∶1, which was allcis in the Control Diet, was replaced bytFA in the Trans Diet. At selected ages, body weight, epididymal fat pad weight, perirenal fat yield, adipose tissue cellularity and fatty acid composition were examined. Over the time period studied (2-24 mon), the proportion of 18∶0 and 16∶0 tended to decrease whilecis-18∶1 levels increased. Compared to the Control Diet, the Trans Diet resulted in adipose tissue lipids with higher percentages of 14∶0 and 18∶2n-6 and lower percentages ofcis-18∶1 and 20∶4n-6. In polar lipids,tFA replaced saturated fatty acids, whereastFA replacedcis-18∶1 in the nonpolar lipids. Body weights at 16 and 24 mon of age and epididymal fat pad weights at 8-24 mon of age were lower in mice fed the Trans Diet as compared to those fed the Control Diet. At the ages studied, the Trans Diet also resulted in lower values for perirenal fat weights, triacylglycerol to polar lipid ratios, and adipose cell size. The data suggest that chronic consumption oftFA affects lipid metabolism and results in decreased fat accumulation in murine adipose tissue.

Effects of trans fatty acids on lipid accumulation in 3T3-L1 cells.

Previous work had shown that dietary trans fatty acids (tFA) resulted in decreased fat deposition in adipose tissue. This study was conducted to see if tFA influence lipid accumulation in Swiss mouse fibroblast 3T3-L1 cells, which are widely used as an adipocyte model. Cells were cultured in the presence of experimental or control growth media supplemented with fatty acids complexed to bovine serum albumin. Fatty acid compositions of experimental and control growth media were similar except that the octadecenoates in the control growth media were cis fatty acids, whereas those in the experimental media contained both cis and trans fatty acids. Cell-conditioned media and cellular lipids at the preadipocyte and differentiating adipocyte stages were analyzed. At both stages of development, less fat accumulated in cells cultured in the presence of tFA, due primarily to a decrease in the nonpolar lipid content of cells exposed to tFA, and linoleate to arachidonate ratios were higher in cells supplemented with tFA. Calculations comparing sums of saturated and monounsaturated fatty acids in cells at the differentiating adipocyte stage suggested that tFA may have replaced monounsaturated fatty acids in the nonpolar lipid fraction and saturated fatty acids in the polar lipid fraction. The results of these studies are in good agreement with the in vivo effects of tFA seen in previous work with mouse adipose tissue. It was concluded that the 3T3-L1 in vitro model is an appropriate system for further studies of tFA and lipid metabolism in adipose tissue.

Positional specificity of gastric hydrolysis of long-chain n-3 polyunsaturated fatty acids of seal milk triglycerides.

Long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) of marine oils are important dietary components for both infants and adults, and are incorporated into milks following maternal dietary intake. However, little is known about the hydrolysis of these PUFA from milk triglycerides (TG) by lipases in suckling young. Seals, like humans, possess gastric lipase; however, the milk lipids of seals and sea lions are almost devoid of the readily hydrolyzable medium-chain fatty acids, and are characterized by a large percentage (10-30%) of n-3 PUFA. Gastric hydrolysis of milk lipids was studied in vivo in suckling pups of three species (the California sea lion, the harp seal and the hooded seal) in order to elucidate the actions and specificity of gastric lipases on milk TG in relation to fatty acid composition and TG structure. Regardless of milk fat content (31-61% fat) or extent of gastric hydrolysis (10-56%), the same fatty acids were preferentially released in all three species, as determined by their relative enrichment in the free fatty acid (FFA) fraction. In addition to 16:1 and 18:0, these were the PUFA of 18 carbons and longer, except for 22:6n-3. Levels of 20:5n-3 were most notably enriched in FFA, at up to five times that found in the TG. Although 22:6n-3 was apparently also released from the TG (reduced in the diglyceride), it was also notably reduced in FFA.(ABSTRACT TRUNCATED AT 250 WORDS)

Lactation curves and effect of pup removal on milk fat of C57Bl/6J mice fed different diet fats.

Groups of C57Bl/6J mice, fed either a cis (C-Diet) or trans diet (T-Diet) were milked without preconditioning at 6, 8, 10 and 12 days postpartum. On day 10, groups of mice were also milked 4, 6 and 18 h after separation of the pups. Except for the 18-h separation, all T-Diet fed animals produced milk of lower fat content than did the C-Diet animals (P < 0.001) throughout the lactation period measured. In the C-Diet mice, the 6-h separation period resulted in a decrease (P < or = 0.03) in fat, but the diet-depressed milk fat of T-Diet animals was not decreased further until the 18-h separation period. Milk volume increased as lactation progressed and was greatly increased as a result of preconditioning (P < or = 0.001), even at 4 h of separation when fat was not reduced, and was always greater for T-Diet animals. Within diet groups, fatty acid composition was similar throughout the lactation period studied and was not affected by preconditioning except in the 18-h separation period, when de novo fatty acids were significantly reduced (P < or = 0.05). The data are consistent with the hypothesis that preconditioning results in lowered milk fat values and that preconditioning techniques can explain discrepancies in literature values for murine milk fat.

Isomeric trans fatty acids in the U.S. diet.

Since actual consumption data for trans fatty acid (FA) intakes for the U.S. population do not exist, estimates of trans fatty acids (FAs) available in the U.S. food supply have been calculated from U.S. Department of Agriculture-Economic Research Service (USDA-ERS) fats and oils production figures and food disappearance data for fats and oils. Based on weighted averages for the trans levels in each fats and oils category, these estimates of trans FAs available in the U.S. food supply range from 12.5 to 15.2 g/person/day (average 13.3 +/- 1.1 g/person/day). Estimates of trans FA consumption have been calculated; these estimates predict a wide range from 1.6 to 38.7 g/person/day. These calculations are based on published estimates of trans FAs available in the total fat of 5-15%, and the total fat intake (range 31-258 g/person/day) of a representative sample of adults (ages 20-59) as determined by the Lipid Research Clinics (LRC). Using an equation based on a relationship between trans FAs in adipose tissue and dietary fat, an intake range of 0.7-28.7 g/person/day trans FAs for the same LRC fat consumption data can be predicted. Adipose tissue isomer profiles that indicate 90-95% of the trans FAs in the tissues comes from partially hydrogenated vegetable fats and oils allow us to predict a dietary intake range from 11.1 to 27.6 g/person/day trans FAs. The significance of these estimates to nutrition policy is discussed.

Milk fat depression in C57Bl/6J mice consuming partially hydrogenated fat.

Mice of the C57Bl/6J strain were maintained on diets in which the unsaturated fatty acids were all cis fatty acids (CFA) or a mixture of CFA and trans fatty acids (TFA). The fats used were mixtures of corn oil, olive oil, cocoa butter, margarine and shortening blended to yield similar fatty acid compositions, except for the ratio of the CFA to TFA and the percentage of linoleic acid (EFA). Regardless of the level of fat (20 or 40 energy %) or the level of EFA (2 to 12 energy %), diets with TFA decreased the percentage of fat in mouse milk. When lactating females raised on the CFA diets were crossed to the TFA diets, TFA appeared in the milk at 12 h postcross, and within 4 d postcross the percent of milk fat was decreased to levels similar to that of nursing females raised continuously on the TFA diets. Conversely, lactating females crossed from TFA to CFA diets produced milk with percentage fat values and fatty acid compositions that approached those seen in nursing females fed the CFA diets continuously. The possible involvement of TFA in the classical milk fat depression phenomenon in ruminants and its potential relevance in human lactation are discussed.

Dietary trans fatty acids modulate erythrocyte membrane fatty acyl composition and insulin binding in monkeys.

The substitution of trans- for half of the cis-monounsaturated fatty acids in the diet of Macaca fasicularis monkeys resulted in alterations in erythrocyte fatty acid composition and insulin receptor properties but not in membrane fluidity. Both cis and trans diets contained 10% fat and similar fatty acid compositions, except that approximately 50% of the cis-octadecenoate (c-18:1) in the cis diet was replaced with trans-octadecenoate isomers (t-18:1) in the trans diet. Compared with the cis diet, the trans diet resulted in the incorporation of approximately 11% t-18:1, an approximately 50% decrease in c-18:1, an approximately 16% decrease in total saturated fatty acids, and an approximately 20% increase in 18:2(n-6) in erythrocyte membrane lipids. The increase in 18:2(n-6) may reflect on homeostatic mechanisms designed to maintain overall membrane fluidity, as no diet-related changes in fluidity were observed with diphenylhexatriene steady state fluorescence polarization. Values observed for insulin binding and insulin receptor number were higher and binding affinity was lower in monkeys fed the cis diet. In the absence of an effect on overall membrane fluidity, altered receptor activity suggests that insulin receptor activity is dynamic, requiring specific fluid membrane subdomains or highly specific fatty acid-protein interactions.

Cheek cell fatty acids as indicators of dietary lipids in humans.

Analysis of cheek cell lipids has been suggested as a noninvasive method for monitoring the fatty acid composition of diets in humans. In a pilot study conducted to determine the validity of the method, cheek cell samples were collected from subjects consuming a low fat (20% of calories) diet consisting of fatty acids with either a 1.0 or 0.3 P/S ratio. Neither total lipid nor polar lipid fatty acids in cheek cells consistently reflected the P/S ratio of the diets. However, there were trends, particularly in the nonpolar lipids, suggesting that cheek cell fatty acid ratios might be useful for monitoring the fatty acid composition of the diets. The diet with the higher P/S ratio (1.0 vs 0.3) consistently resulted in cheek cell lipids with lower ratios of 18:1/saturated fatty acids and greater 18:2/20:4, 18:2/18:1 and 18:2/18:0 fatty acid ratios.

Analysis of positional isomers of monounsaturated fatty acids by high performance liquid chromatography of 2,4-dinitrophenylhydrazones of reduced ozonides.

A method is described for quantifying the positional isomers in monounsaturated fatty acid methyl ester (FAME) fractions. The procedure involves the preparation of 2,4-dinitrophenylhydrazones (DNPH) of the fragments generated during reductive ozonolysis of FAME, class isolation of the aldehyde and aldehyde ester DNPH, and separation of the aldehyde ester derivatives by high performance liquid chromatography (HPLC). The high extinction coefficient of the DNPH provides for a sensitive assay which is linear for a large range of components over a concentration range of 0.075-5 nmol/component, and the stability of the DNPH permits the independent analysis of the aldehyde and aldehyde ester fragments generated during reductive ozonolysis. The reductive ozonolysis-DNPH-HPLC method developed is as sensitive, reproducible, and accurate as reductive ozonolysis-gas-liquid chromatography and does not suffer from some of the drawbacks of the classical procedure.

Myelin subfractions isolated from mouse brain: analysis of the lipid composition at three developmental stages.

Lipids were examined in whole myelin and 8 myelin subfractions isolated from mouse brain at 18-24, 44-48 and 80-90 days of age. Relative to protein, total lipid was lowest in whole myelin isolated from the oldest animals as well as from subfractions isolated at greater sucrose densities, thus partially accounting for the observed myelin subfraction distribution pattern which shifted during development and an average peak density between 0.55 and 0.65 M sucrose to one banding between 0.60 and 0.70 M sucrose. Whole myelin and each myelin subfraction isolated at one age contained nearly the same ratio of sterol and phospholipid to galactolipid; these ratios decreased uniformly during development suggesting enrichment with galactolipid in all myelin subfractions. Sulfatide, as percentage of total galactolipid, was relatively constant during development and appeared to be slightly enriched in the denser myelin subfractions. The findings suggest that regardless of the origin(s) of the subfractions, an age-related mechanism exists in the central nervous system which modified myelin lipid composition relatively uniformly.

Effects of prolonged starvation on plasma free fatty acid levels and fatty acid composition of myocardial total lipids in the rat.

Experiments were carried out on male rat fed ad libitum or starved for a period of 7 days. Plasma levels of free fatty acids (FFA) and the quantity and composition of fatty acids in total lipids of heart ventricular tissue in vivo were analyzed by gas liquid chromatography (GLC). In addition, FFA extraction ratios and uptake rates were determined in isolated perfused hearts using the classical Langendorff technique. After 7 days of starvation, distribution and concentrations obtained for total lipid fatty acids from heart ventricles of starved animals were substantially different from those of controls. In particular, 20:5 and 22:6 carbon polyunsaturated fatty acids were significantly elevated. Also, total plasma FFA level was elevated and FFA extraction ratios were increased as a result of prolonged starvation. Accumulation of 20- and 22-carbon polyunsaturated fatty acids observed in ventricles of starved rats may have resulted from increased availability and extraction of FFA.

Laser-Raman investigation of phospholipid-polypeptide interactions in model membranes.

The interaction of aqueous dimyristoyl-phosphatidylcholine liposomes with the polypeptides gramicidin A, poly-L-lysine, valinomycin, and gramicidin S was investigated by means of laser-Raman spectroscopy. Auxiliary data were obtained with differential scanning calorimetry. Studies were carried out over the temperature range of 0--50 degrees C, encompassing the gel phase, the transition region, and the liquid crystalline phase of the liposomes. Conformational changes in the phospholipid molecules were investigated by measuring the intensity of the 1062-cm-1 Raman band which is assigned to C-C stretching vibrations of trans segments. Three different types of phospholipid-polypeptide interactions were indicated by the observed Raman data. They are interpreted as (a) orderly penetration of the phospholipid bilayer by a hydrophobic polypeptide; (b) polar interactions involving primarily the head groups of the phospholipid; and (c) disorderly hydrophobic binding between a polypeptide and the hydrocarbon domain of the phospholipid.

Myelin subfractions isolated from mouse brain. Studies of normal mice during development, quaking mutants, and three brain regions.

Myelin isolated from three areas of mouse brain, from whole brain at several ages in normal mice, and from whole brain of adult quaking mutant mice was separated into seven bands and a pellet on discontinuous density gradients using 0.32, 0.45, 0.55, 0.60, 0.70, 0.75 and 0.85 M sucrose. The distribution of myelin in the subfractions was independent of homogenization and shocking conditions employed to isolate the myelin preparations, but was related to the type of myelin applied to the gradient. Compared to myelin isolated from older animals, myelin isolated from 18-24 day old mice displayed a distribution pattern with greater proportions of material banding at lesser sucrose densities. Similarly, myelin obtained from hindbrain contained proportionately more material layering at lesser sucrose densities compared to myelin isolated from cerebral cortex. Myelin subfraction patterns observed for 8-12 day old control mice and quaking mutants were unlike each other or any other myelin preparation examined. In the 18-90 days old animals, the markers studied were not uniformly distributed among the myelin subfractions. The pellet and the layer banding at the 0.75/0.85 M sucrose interface contained the highest specific concentrations of sialic acid, nucleic acid, and total adenosine triphosphatase activity. In contrast, the specific activity of 2',3'-cyclicnucleotide-3'-phosphohydrolase was lowest in the pellet as well as the three bands obtained above 0.60 M sucrose and was highest in the fraction banding at the 0.65/0.70 M sucrose interface. The results obtained were not consistent with an artifactual origin of the myelin subfractions, but instead suggested that the subfraction have physiological significance. One explanation for the different banding patterns observed between young and mature myelin may be the different amount of myelin in various brain regions during development.