Influence of linoleic acid on desaturation and uptake of deuterium-labeled palmitic and stearic acids in humans.

Objectives of this study were to investigate the desaturation of stearic acid (18:0) and palmitic acid (16:0), to determine if differences in their metabolism provide a reasonable explantation for differences in their effect on serum cholesterol levels, and to investigate the affect of linoleic acid on delta 9-desaturase products in man. Deuterium-labeled 16:0 and 18:0 were used to follow the metabolism of these fatty acids in young adult male subjects that were pre-fed diets containing two different levels of linoleic acid. Results indicate that absorption of 16:0 and 18:0 was similar when all components of the mixture used to formulate the deuterated fat mixture were kept above the melting point of tristearin. The percent of 18:0 desaturated to 9c-18:1 was higher than the percent of 16:0 desaturated to 9c-16:1 (9.2% vs. 3.9%). The subject-to-subject variability suggests that differences in ability to desaturate saturated fatty acids may be related to the variability observed in response of serum cholesterol levels to dietary saturated fatty acids. Data for the distribution of 16:0 and 18:0 between triacylglycerol and phosphatidylcholine (PC) was markedly different. Based on PC data, phospholipid acyltransferase selectivity was about 2-fold higher for 18:0 than for 16:0. A 2-fold difference in the linoleic acid content of the pre-fed diets had little influence on desaturation or distribution of 16:0 and 18:0 between plasma lipid classes. A deuterium isotope effect was estimated to reduce delta 9-desaturase enzyme activity by 30-50%.

Measurement of the metabolic interconversion of deuterium-labeled fatty acids by gas chromatography/mass spectrometry.

An analytical method that was developed to analyze deuterium-labeled fatty acids in human blood has been extended to identify labeled fatty acids from C14 to C24 chain length which are formed by metabolic processes such as desaturation, elongation, or shortening of the labeled fatty acids fed. A new computer and a hardware adder have been utilized to assure reliable data acquisition. Relative standard deviations for the analysis of labeled fatty acids were measured at 0.02, 0.03, and 0.04 at the 5%, 1%, and 0.2% levels of the labeled fatty acid methyl esters, respectively. The method makes extensive use of standards and computer processing for accuracy and high productivity. Data from a chylomicron triacylglycerol fraction are included to demonstrate the sensitivity of detection of metabolites formed by desaturation and elongation.

Incorporation of trans-8- and cis-8-octadecenoic acid isomers in human plasma and lipoprotein lipids.

Mixtures of deuterium-labeled trans-8, cis-8 and cis-9-octadecenoic acids (8t-18:1, 8c-18:1, 9c-18:1) were fed as triglycerides (TG) to two adult male subjects. Blood samples were collected sequentially over a 48-hour period. Plasma and lipoprotein lipids were separated by thin layer chromatography and analyzed by gas chromatography-mass spectroscopy. Results indicate (i) absorption of the 8t- and 8c-18:1 isomers were similar to 9c-18:1; (ii) the 8t-18:1 isomer was cleared approximately 30% faster than 9c-18:1 from plasma TG; (iii) cholesterol ester samples contained 8.4 times less 8t-18:1 than 9c-18:1; (iv) incorporation at the 1-acyl phosphatidylcholine (PC) position was higher for 8t-18:1 and 8c-18:1 (2.2 and 1.7 times) than for 9c-18:1; and (v) discrimination at the 2-acyl PC position was 4.6-fold against 8t-18:1 and 1.3-fold against 8c-18:1 compared with 9c-18:1. Discrimination against uptake of the delta-8 isomers in both neutral and phospholipid classes suggests that both 8t- and 8c-18:1 may be preferentially oxidized relative to 9c-18:1. Except for triglycerides, data for each of the lipid classes from total plasma and individual lipoprotein samples were similar. These data indicate that differences for incorporation and turnover of the 8t- and 8c-18:1 isomers relative to 9c-18:1 are not substantially influenced by the lipoprotein classes. The maximum isotopic enrichment detected in the chylomicron triglycerides fractions was 60%, which indicates that a substantial amount of endogenous triglycerides was mobilized during absorption of the deuterated fats.

Metabolism in humans of cis-12,trans-15-octadecadienoic acid relative to palmitic, stearic, oleic and linoleic acids.

Mixtures of triglycerides containing deuterium-labeled hexadecanoic acid (16:0), octadecanoic acid (18:0), cis-9-octadecenoic acid (9c-18:1), cis-9,cis-12-octadecadienoic acid (9c, 12c-18:2) and cis-12,trans-15-octadecadienoic acid (12c,15t-18:2) were fed to two young-adult males. Plasma lipid classes were isolated from samples collected periodically over 48 hr. Incorporation and turnover of the deuterium-labeled fats in plasma lipids were followed by gas chromatography-mass spectrometry (GC-MS) analysis of the methyl ester derivatives. Absorption of the deuterated fats was followed by GC-MS analysis of chylomicron triglycerides isolated by ultracentrifugation. Results were the following: (i) endogenous fat contributed about 40% of the total fat incorporated into chylomicron triglycerides; (ii) elongation, desaturation and chain-shortened products from the deuterated fats were not detected; (iii) the polyunsaturated isomer 12c,15t-18:2 was metabolically more similar to saturated and 9c-18:1 fatty acids than to 9c,12c-18:2; (iv) relative incorporation of 9c,12c-18:2 into phospholipids did not increase proportionally with an increase of 9c,12c-18:2 in the mixture of deuterated fats fed; (v) absorption of 16:0, 18:0, 9c-18:1, 9c,12c-18:2 and 12c,15t-18:2 were similar; and (vi) data for the 1- and 2-acyl positions of phosphatidylcholine and for cholesteryl ester fractions reflected the known high specificity of phosphatidylcholine acyltransferase and lecithin:cholesteryl acyltransferase for 9c,12c-18:2. These results illustrate that incorporation of dietary fatty acids into human plasma lipid classes is selectively controlled and that incorporation of dietary 9c,12c-18:2 is limited. These results suggest that nutritional benefits of diets high in 9c,12c-18:2 may be of little value to normal subjects and that the 12c,15t-18:2 isomer in hydrogenated fat is not a nutritional liability at the present dietary level.

Absorption and distribution of deuterium-labeled trans- and cis-11-octadecenoic acid in human plasma and lipoprotein lipids.

Triglycerides of deuterium-labeled trans-11-, trans-11-cis-11- and cis-9-octadecenoic acid (11t-18:1-2H, 11c-18:1-2H) were simultaneously fed to two young adult male subjects. Plasma lipids from blood samples collected periodically for 48 hr were analyzed by gas chromatography-mass spectroscopy. The results indicate the delta 11-18:1-2H acids and 9c-18:1-2H were equally well absorbed; relative turnover rates were higher for the delta 11-18-1-2H acids in plasma triglycerides; incorporation of the delta 11-18:1-2H acids into plasma phosphatidylcholine was similar to 9c-18:1-2H, but distribution at the 1- and 2-acyl positions was substantially different; esterification of cholesterol with 11t-18:1 was extremely low; chain shortening of the delta 11-18:1-2H acids was 2-3 times greater than for 9c-18:1-2H; no evidence for desaturation or elongation of the 18:1-2H acids was detected; and a 40% isotopic dilution of the 18:1-2H acids in the chylomicron triglyceride fraction indicated the presence of a substantial intestinal triglyceride pool. Based on our present knowledge, these metabolic results for delta 11-18:1 acids present in hydrogenated oils and animal fats indicate that the delta 11 isomers are no more likely than 9c-18:1 to contribute to dietary fat-related health problems.

In vivo distribution and turnover of trans- and cis-10-octadecenoic acid isomers in human plasma lipids.

Triacylglycerols containing deuterium-labeled trans-10- and cis-10-octadecenoic acid (10t-18:1, 10c-18:1) plus the triacylglycerol of deuterated cis-9-octadecenoic acid (9c-18:1) were fed as a mixture to two young, adult male subjects. Analysis by mass spectroscopy of the labeled fats in blood samples collected periodically for 48 h allowed the uptake, distribution and turnover of both 10-octadecenoic acid isomers to be directly compared to 9c-18:1. A feature of this study is that actual weight data for the labeled fats were obtained. These data allowed plasma triacylglycerol turnover rates of 3.47-5.13 mg/min per kg to be estimated. Plasma and chylomicron triacylglycerol data also provided evidence that absorption of the deuterated fats mobilized 10-12 g of a triacylglycerol pool present in the intestinal cells. Other results are summarized as follows: the 10t-, 10c- and 9c-18:1 fatty acids were equally well absorbed, both delta 10-18:1 isomers were oxidized more rapidly than 9c-18:1, conversion of the delta 10-18:1 isomers into their corresponding 16:1 isomers was about 3-times faster than for 9c-18:1, the delta 10-18:1 isomers were preferentially incorporated at the 1-acyl and excluded from the 2-acyl position of phosphatidylcholine, esterification of cholesterol with the delta 10-18:1 fatty acids was 2.5-4.3-times slower than for 9c-18:1 and desaturation and elongation rates for the delta 10-18:1 acids were very low.

Analysis of deuterium labeled blood lipids by chemical ionization mass spectrometry.

A quantitative analytical method has been developed to analyze methyl esters of blood fatty acids derived from human subjects fed deuterium-labeled fats. The GCMS computer method provides for the analysis of the fed deuterium-labeled fatty acids, the naturally occurring blood fatty acids and new fatty acids formed by chain elongation or shortening of the fed labeled fats. Approximately 20 fatty acids including 16, 17, 18 and 20 carbon chain acids were analyzed with a relative standard deviation of 0.02 at the microgram level and a sensitivity of less than one nanogram. The method uses capillary GC to separate the fatty acid esters and isobutane chemical ionization mass spectrometry with multiple ion detection to determine the isotopic constituents of the GC peaks. The technique provides for the determination of overlapping GC peaks labeled with 2, 4 and 6 deuterium atoms and makes extensive use of computers both for data acquisition and processing.

Incorporation of deuterium-labeled trans- and cis-13-octadecenoic acids in human plasma lipids.

The absorption and distribution of deuterated trans- and cis-13-octadecenoic acid (13t-18:1 and 13c-18:1) in plasma lipids were compared to deuterated cis-9-octadecenoic acid (9c-18:1) in two young adult male subjects. A mixture of triglycerides was fed in a multiple-labeled experiment where each triglyceride contained a fatty acid labeled with a different number of deuterium atoms. Analysis of human plasma lipids by mass spectroscopy allowed the distribution of the two 13-octadecenoic acid isomers to be directly compared to cis-9-octadecenoic acid. Plasma lipids selectively excluded both the 13t-18:1 and 13c-18:1 isomers relative to 9c-18:1 in all neutral and phospholipid fractions. Discrimination against incorporation of the 13t-18:1 isomer into plasma cholesteryl ester and 2-acyl phosphatidylcholine was nearly absolute. The 1-acyl phosphatidylcholine fraction exhibited a large positive selectivity for the 13t-18:1 isomer. Differences in the relative distribution of the trans and cis 13-18:1 isomers vs. 9c-18:1 in the various lipoprotein lipid classes were found. Analysis of the chylomicron triglyceride component of the plasma lipids indicated all three fatty acids were equally well absorbed.

Experimental trichothecene mycotoxicosis produced in broiler chickens by Fusarium sporotrichiella var. sporotrichioides.

Fusarium sporotrichiella var. sporotrichioides (Bilay), cultured on . sterilised popcorn at 23 degrees C and then at 8 degrees C, 16 degrees C and 23 degrees C and fed as 50% of the diet, was lethal to 7-day-old male broiler chickens. The 8 degrees C culture, containing T-2 toxin at 50 parts per million (ppm) and neosolaniol at 5 ppm, was given as whole culture at dietary concentrations of 10%, 5%, 1% and 0% for 17 days and 1% for 42 days. Half the chickens that were fed the 10% diet died during the 17 days (5 ppm T-2 toxin and 0.5 ppm neosolaniol). The corresponding daily dose was 0.24 mg T-2 toxin and 0.02 mg neosolaniol/kg body weight/day. The chickens that died were dehydrated, had necrosis and depletion of lymphoid and haematopoietic tissues and necrosis of the hepatobiliary system, gastroenteric mucosa, feather epidermis and renal tubular epithelium. The survivors had anaemia, reduction of weight gain and transiently altered righting reflex. The comb and beak were pale yellow and the feather barbs were dishevelled. Survivors also had atrophied lymphoid tissues, reduced haematopoietic cellularity in the bone marrow, necrosis of oral and crop mucosa, vacuolated hepatocytes, hyperplastic bile ductules, and reduction of the thyroid follicular diameter.

Elaboration of vomitoxin and zearalenone by Fusarium isolates and the biological activity of Fusarium-produced toxins.

Sixteen Fusarium isolates belonging to F. graminearium Schw. and F. culmorum (W.G. Smith) Sacc. produced vomitoxin and zearalenone on cracked corn at 28 degrees C. Quantitation for vomitoxin was by gas-liquid chromatography. This toxin was produced in quantities of 5 to 236 microgram/g of fermented corn. Vomitoxin showed weak antibiotic activity against Penicillium digitatum Sacc., Mucor ramannianus Möller, and Saccharomyces bayanus Sacc., but did not inhibit gram-positive, gram-negative, or acid fast bacteria. The two molds and the yeast were inhibited by T-2 toxin at 5 micrograms, and diacetoxyscirpenol inhibited the molds at 5 micrograms and the yeast at 50 micrograms.

Swine refusal factors elaborated by Fusarium strains and identified as trichothecenes.

Fusarium poae (Peck) Wollenw. NRRL 3287, F. nivale (Fr.) Ces. NRRL 3289, and F. moniliforme Sheldon NRRL 3197, each grown on cracked corn (13 days at 28 degrees C), produced refusal factors in pig bioassays. Substantial quantities of trichothecenes were detected in the refused corn: T-2 toxin (30 micrograms/g) was detected in corn fermented with the F. poae strain; the level of vomitoxin (1 microgram/g) in corn cultured with F. nivale did not account for the 48% refusal response in the pigs tested. The F. moniliforme concomitantly produced T-2 toxin (33 micrograms/g) and vomitoxin (1.5 micrograms/g). This strain's taxonomic position was reexamined, and it is shown to be a cultural variant of the species F. tricinctum (Cda.) Sacc.

Distribution of deuterium-labeled cis- and trans-12-octadecenoic acids in human plasma and lipoprotein lipids.

Triglycerides containing cis- and trans-12-octadecenoic acid (12c-18:1 and 12t-18:1) and cis-9-octadecenoic acid (9c-18:1) labeled with deuterium were fed to 2 young adult male subjects. These fatty isomers each contained a different number of deuterium lables, which allowed mass spectrometric analysis to distinguish among them after they were fed as a mixture. This approach results in a direct comparison of the absorption and distribution of these 3 monenoic acids into blood plasma and lipoprotein lipids. Plasma lipid data indicated that all phospholipid fractions selectively incorporate 12c-18:1 and 12t-18:1 in preference to 9c-18:1. Discrimination against 12c-18:1 and 12t-18:1 compared to 9c-18:1 was found in the plasma neutral lipids, with a strong discrimination against 12t-18:1 incorporation into the cholesteryl ester fraction. Considerable reduction in the percentage of linoleic and arachidonic acid was observed when 12-18:1 isomers were incorporated in plasma triglyceride, phosphatidylcholine and spingomyelin samples. Chylomicron lipid analyses indicated that all isomers were well absorbed. Variation was observed in the relative distribution of 12c-18:1, 12t-18:1 and 9c-18:1 between the very low density, low density and high density lipoprotein lipid classes. No desaturation of 12c-18:1 to linoleic acid was detected.

Incorporation of deuterium-labeled cis- and trans-9-octadecenoic acids in humans: plasma, erythrocyte, and platelet phospholipids.

The objective of this study was to follow the uptake and distribution of oleic and elaidic acids into human erythrocytes, platelets, and plasma phospholipids. The use of dual and triple labeling methodology permitted a precise comparison of elaidic and oleic acid utilization. Elaidic acid (EI) was selectively concentrated in all the plasma phospholipids except for lysophosphatidylcholine. Three times more elaidic than oleic acid (OI) accumulated in the 1-acyl position of phosphatidylcholine, as determined by hydrolysis with phospholipase A2. Rapid incorporation and removal of elaidate were observed for all samples. These results support the concept that enzymes responsible for acylation of phospholipids are sensitive to double bond configuration and the physical properties of the fatty acid moieties. Labeled fatty acid levels in red cell and platelet phospholipids were much lower than for plasma phospholipids, indicating a relatively slow rate for the in vivo incorporation of fatty acids into blood cell membrane phospholipids. No isotope effect was found when oleic acid labeled with deuterium on the double bond was used.

Mass spectrometric analysis of deuterium dual labeled blood lipids.

A gas chromatography selected ion monitoring mass spectrometer technique was developed to analyze deuterium dual labeled blood lipid samples from a human feeding experiment. In the metabolism experiment, described elsewhere. [2H2] labeled cis and [2H4] labeled trans fatty acids were fed to a human sujbect as a single pulse in order to determine whether the human body differentiates between cis and trans fatty acids in the diet. The analytical method described here was developed to accurately measure the ratio of [2H2]methyl oleate to [2H4]methyl elaidate in the presence of large amounts of [2H0] methyl oleate in samples derived from separated fractions of blood lipids. The technique is different from most selected ion monitoring methods in that the internal standard was fed to the subject along with the experimental material; the samples contained large amounts of unlabeled material chemically identical to the labeled material and the ratio of [2H2] to [2H4] fatty esters was of principal interest rather than the absolute value. Nine standards were analyzed eight or more times to form a basis for statistical evaluation of the method. Analysis of the plasma phosphatidyl ethanolamine fraction from the metabolic experiment is given as an example.