Rat testicular lipids and dietary isomeric fatty acids in essential fatty acid deficiency

Weanling rats were fed essential fatty acid-deficient diets, either completely fat-free, or with partially hydrogenated fish oil (PHFO, 28 wt %), or with fractions derived from PHFO containing primarily positional isomers oftrans-eicosenoate (20∶1, 3 wt %) ortrans-docosenoate (22∶1, 3 wt %). Control animals were fed a peanut oil-containing diet (28 wt %). After 5 or 15 weeks on the diet, the content of neutral and phosphorus-containing lipids in the testes was determined. The fatty acid distribution in major lipid classes was analyzed for animals fed the diets for 15 weeks. The testicular stage of maturation or degeneration was assessed by histology. The group fed PHFO exhibited signs of complete testicular degeneration, or lack of maturation, already after 5 weeks, whereas the animals on the diets with the very long chain monoenoic acids suffered severe degenerations only after 15 weeks. In the PHFO-fed rats, a sharp decline in the concentration of testicular triacylglycerols was observed. In all of the essential fatty acid-deficient groups, an increase in testicular sphingomyelin was observed. Cholesterol levels were fairly similar among all dietary groups. The total testicular fatty acids of the PHFO-fed animals contained somewhat more eicosadienoic acid than found in the other groups, and somewhat less (n−9)-acids. In all EFA-deficient groups, (n−6)-acids were lowered, in particular in triacylglycerols and phosphatidyl cholines. The PHFO group did not show a lower (n−6)-concentration than the other deficient groups, in spite of the more severe symptoms of deficiency. There was no evidence of a major accumulation of long chain isomeric fatty acids in the degenerated testes of the PHFO-, 20∶1, and 22∶1-fed groups.     

Trans octadecenoic fatty acid (TFA) isomers in German foods and bakery goods

In the present study, 122 food samples from the German food market were analysed for their C18:1 trans fatty acid (TFA) content and profile. A particular focus of the survey were baked and fried foods. TFA analysis was performed by means of silver ion SPE (Ag⁺‐SPE) in combination with high‐resolution GC (HRGC‐FID). Overall, 51 bakery product samples were analysed of which 25 samples were prepacked bakery products purchased from local retail stores and 26 samples of unpacked bakery products purchased from local bakery shops. In addition, 14 French fries samples obtained from small local fast food restaurants as well as from internationally operating fast food chains, 27 potato and tortillas chips, 15 instant soups as well as 15 dry culinary sauces were analysed. The highest amounts of C18:1 TFA isomers were found in deep‐fried bakery products. Prepacked branded cookies and biscuits on the other hand contained only negligible C18:1 TFA amounts. Regarding their C18:1 trans isomer profile most deep‐fried bakery products exhibited a Gaussian‐distributed isomer profile. The analysed prepacked croissants, cookies and biscuits contained predominantly ruminant TFA (TFA) as suggested by the presence of vaccenic acid (C18:1 trans 11), which was the major C18:1 TFA isomer in these products. All non‐bakery samples (n = 71) contained less than 3 g C18:1 TFA per 100 g fat. In conclusion, TFA still occur in considerable amounts in a few German food products, especially in some deep‐fried bakery products (‘Berliner’ type of doughnuts). Practical applications: Trans fatty acids, in particular the trans octadecenoic fatty acid isomers (C18:1), are generally considered from the nutritional point of view as undesirable food components due to their negative health effects. Tremendous efforts have been made by major food processors in order to decrease or even eliminate the presence of TFA in some foodstuffs (e.g. in margarines in European countries). However, some food processors of other food sectors are still applying oils and fats containing partially hydrogenated vegetable oils, whereas others within the same food category have already switched their processing conditions and/or raw materials towards TFA alternatives. Therefore, actual TFA data of foodstuffs determined by means of state‐of‐the‐art analytical procedures (Ag⁺‐SPE in combination with GC‐FID) is necessary to detect areas of further improvement in the food supply chain and to provide data for an update of dietary TFA intake.     

Distribution of hexadecenoic,octadecenoic and octadecadienoic acid isomers in human tissue lipids

Thetrans 16∶1, 18∶1 and 18∶2 fatty acid composition of various human organ lipids was studied to determine if isomers accumulated in specific tissues. “Trans” isomers are defined as those fatty acids containing one or moretrans double bonds. Adipose, kidney, brain, heart and liver tissue lipids were analyzed. Gas chromatography with a 100-SP2560 capillary column was used to characterize the various positional and/or geometrical isomers. The distribution ofrans 16∶1 and 18∶1 isomers ranged from 0.3% in the brain to 4.0% in adipose tissue, whiletrans 18∶2 isomers ranged from 0.0% in the brain to 0.4% in adipose tissue. Notrans 18∶3 isomers were detected. Positional isomer ratios forcis 16∶1 (Δ9 vs Δ7) andcis 18∶1 (Δ11 vs Δ9) were also determined. Since these ratios are reproducible from one individual to the next, they might be useful for diagnosis of human metabolic disorders.     

The in vivo incorporation of labeled linoleic acid, α-linolenic acid and arachidonic acid into rat liver lipids

The incorporation of 1-¹⁴C-linoleic acid, 1-¹⁴C-α-linolenic acid and 1-¹⁴C-arachidonic acid into rat liver lipids was measured and the per cent distribution of radioactivity into the different lipid fractions determined. Normal rats were injected into the portal vein with the labeled solutions during a one minute period. Livers were quickly frozen, pulverized, and the lipids extracted and fractioned by thin layer chromatography. No significant differences were observed in the amounts of labeled fatty acids incorporated per gram of rat liver. While 1-¹⁴C-linoleic acid and 1-¹⁴C-α-linolenic acid were found in appreciable amounts in the 1,2 diacylglycerol fraction, about one fifth as much 1-¹⁴C-arachidonic acid was esterified in this fraction. 1-¹⁴C-arachidonic acid was the leading acid esterified in the phospholipid fractions.     

Dietary arachidonic acid and hepatic desaturation of fatty acids in obese zucker rats

The effect of low levels of dietary arachidonic acid (20:4n-6) on Δ6 desaturation of linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3), and on Δ5 desaturation of dihomo-γ-linolenic acid (20:3n-6) were studied in liver microsomes of obese Zucker rats, in comparison with their lean littermates. Fatty acid composition of serum total lipids and of phospholipids from liver microsomes and from total heart and kidney was determined to see whether modifications of desaturation rate, if any, were reflected in the tissue fatty acid profiles. Animals fed for 12 wk on a balanced diet, containing 20:4n-6 and 18:2n-6, were compared to those fed 18:2n-6 only. The low amount of dietary 20:4n-6 greatly inhibited Δ6 desaturation of 18:2n-6 and Δ5 desaturation of 20:3n-6, whereas Δ6 desaturation of 18:3n-3 was slightly increased in obese rats. Inhibition of the biosynthesis of long-chain n-6 fatty acids by dietary arachidonic acid was only slightly reflected in the 20:4n-6 content of liver microsome phospholipids. On the contrary, the enrichment of serum total lipids and heart and kidney phospholipids in this fatty acid was pronounced, more in obese than in lean animals. Our results show that, although the desaturation rate of the n-6 fatty acids in liver microsomes was greatly decreased by the presence of arachidonic acid in the diet, the tissue phospholipid content in arachidonic acid was not depressed. The potentiality of synthesis of eicosanoids of the 2 family from this fatty acid is consequently not lower, especially in obese rats, in which certain tissues are deficient in arachidonic acid, in comparison with their lean littermates.     

Availability of arachidonic acid in major phospholipids of mucosa and the stomach wall of rats

The purpose of this study was to examine the availability of arachidonic acid in phosphatdidylcholine (PC) and phosphatidylethanolamine (PE) of mucosa and the stomach wall of adult male Wistar rats fed a standard diet. There were significant differences in the fatty acid composition of PC and PE between various parts of the stomach. The mucosa had the lowest level of arachidonic acid (20:4n-6) in PC, followed by the corresponding stomach wall or glandular part, whereas the forestomach or the upper part had the highest level of 20:4n-6. The level of 20:4n-6 in PE was identical in gastric mucosa and the stomach wall of both the lower and upper parts. The levels of 18:1n-9 and 18:2n-6 were significantly higher in both PC and PE of mucosa than in the stomach wall. The levels of 16:0 and 18:0 in PC were lower in mucosa than in either forestomach or the glandular part. In mucosal PE, the levels of 18:0 and 22:6n-3 were lower than in forestomach and glandular part. The glandular part had significantly higher level of 16:0 and lower level of 20:4n-6 in PC compared to the forestomach. Conclusions: The arachidonic acid level of PC was significantly different in various parts of the rat stomach, with the lowest level in mucosa, whereas the level of 20:4n-6 in PE was identical in mucosa and stomach wall. These phospholipids had higher levels of saturated fatty acids in the stomach wall than in gastric mucosa.     

Effects of purified eicosapentaenoic acid on arachidonic acid metabolism in cultured murine aortic smooth muscle cells,vessel walls and platelets

The effects of highly purified eicosapentaenoic acid (97% pure) on the arachidonic acid cascade in isolated murine vascular cells and platelets were studied. The incorporation of eicosapentaenoic acid was not as active as that of arachidonic acid in platelets. The ratio of incorporation of eicosapentaenoic acid to arachidonic acid into platelet phospholipids was about 0.7. Analysis of the phospholipid fractions of platelets after labeling with¹⁴C-eicosapentaenoic acid and¹⁴C-arachidonic acid revealed that the incorporation of¹⁴C-eicosapentaenoic acid into the phosphatidylinositol fraction is significantly less than that of¹⁴C-arachidonic acid, while the incorporation of both fatty acids into other phospholipid fractions was almost the same. On the other hand, no significant difference between either fatty acid in incorporation rate, kinetics or distribution in cellular phospholipids was found in cultured aortic smooth muscle cells. Following treatment with eicosapentaenoic acid, cells produced less prostacyclin from endogenous arachidonic acid than did control cells. This was not due to the decrease in fatty acid cyclooxygenase activity, but rather, due to the decrease in arachidonic acid content in cellular phospholipids. In addition, eicosapentaenoic acid was neither converted to prostaglandin I₃ by the vascular cells nor to thromboxane A₃ by platelets. Furthermore, similar results were also obtained by in vivo experiments in which rats were fed with eicosapentaenoic acid enriched diet.     

Hepatoma,host liver,and normal rat liver lipids: Distribution of isomeric monoene fatty acids in individual lipid classes

Monoenoic acid fractions were isolated from phosphatidycholine, phosphatidylethanolamine, triglycerides, and cholesteryl esters of hepatoma 7288CTC, host liver, and normal liver from animals maintained on chow and fat free diets. Hexadecanoate (16:1), octadecenoate (18:1), and eisosenoate (20:1) fractions were analyzed quantitatively for their isomeric composition. The fat free diet had little or no effect relative to the chow diet on the isomeric composition of 16:1, 18:1, and 20:1 from any lipid class in either heptoma, host liver, or normal liver. Host livers were reduced in palmitoleic acid, and oleic and eicos-11-enoic acids were increased relative to normal liver. The 16:1 fraction from triglyceride of normal liver, host liver, and hepatoma contained 90, 80, and 75% palmitoleic acid, respectively. The 20:1 fraction from triglycerides of normal liver, host liver, and hepatoma contained ca. 55, 70, and 60% eicos-11-enoic acid, respectively, with the remainder consisting of eicos-13-enoic acid. The proportion of vaccenic acid in the 18:1 fraction was 60, 50, 20, and 25% for phosphatidylethanolamine, phosphatidylcholine, triglycerides, and cholesteryl esters, respectively, with oleic acid making up the balance. In contrast, all hepatoma lipid classes exhibited the same proportion of oleic (70%) and vaccenic (30%) acids. These data appear to be the first to demonstrate lipid class specificity for isomeric octadecenoic acids in normal liver and the loss of this specificity in a neoplasm.     

Ratio of in vivo incorporation of3H arachidonic acid and14C linoleic acid into liver lipids from normal and diabetic rats

Normal and streptozotocin diabetic rats were injected via the portal vein with a labeled solution containing³H arachidonic acid and¹⁴C linoleic acid (³H/¹⁴C ratio, 0.5) during a 1 min period. Livers were quickly frozen, pulverized, and the lipids extracted and fractioned by thin layer chromatography. The incorporation of³H and¹⁴C into liver lipids was measured and the percentage distribution of radioactivity into the different lipid fractions was determined. The incorporation of¹⁴C linoleic acid and³H arachidonic acid into liver lipids is apparently reduced in rats with severe diabetes. The higher³H/^14C ratio found in the 1,2 diglycerides from diabetic rats may be explained by the apparently smaller incorporation of¹⁴C linoleic acid or by an isotopic dilution attributable to the great availability of this acid in diabetic rats. On the other hand, the higher³H/¹⁴C ratio observed in triglycerides and phospholipids from diabetic rats, due to a relatively large incorporation of³H arachidonic acid into this fraction, may be explained by the affinity of the enzymes involved in their synthesis for some 1,2-diglyceride units. Insulin was unable to correct the changes observed in the diabetic rats.     

Docosahexaenoic acid and arachidonic acid levels are correlated in human milk: Implications for new European infant formula regulations

From February 2022, all infant formula sold in the European Union must contain docosahexaenoic acid (DHA) at ~0.33%–1.14% of total fat with no minimum requirement for arachidonic acid (ARA). This work examines the association between DHA and ARA levels in human milk, the gold standard for infant feeding. Human milk (n = 470) was collected over 12-weeks postpartum from lactating mothers (n = 100) of infants born weighing <1250 g (NCT02137473). Fatty acids were analyzed by gas chromatography. ARA and DHA concentrations were associated in human milk (β = 0.47 [95% confidence interval 0.38–0.56] mol%), including transitional and mature milk, but not colostrum. This remained significant upon adjustment for percentages of other saturated, monounsaturated, n-3, or n-6 fatty acids, day of sample collection, or maternal characteristics (body mass index, ethnicity, education, and income). Infant formulas containing relatively high concentrations of DHA without ARA, as permitted by the new regulations, would not reflect the balance of these fatty acids in human milk.     

Trans fatty acids. 3. Fatty acid composition of the brain and other organs in the newborn piglet

The effects of dietarytrans fatty acids on tissue fatty acid composition were studied in newborn piglets delivered from sows fed partially hydrogenated fish oil (PHFO) (28%trans) or partially hydrogenated soybean oil (PHSBO) (36%trans) in comparison with lard (0%trans) from 3 wk of age and through gestation in Experiment 1, or fed PHFO or “fully” hydrogenated fish oil (HFO) (19%trans) in comparison with coconut oil (CF) (0%trans) with two levels, 1 and 2.7%, of dietary linoleic acid from conception through gestation in Experiment 2. The piglets were sampled immediately after delivery, without having access to mothers' milk. Incorporation oftrans fatty acids into brain PE (phosphatidylethanolamine) were non-detectable or very low (less than 0.1%). The incorporation of 18∶1trans into heart-PE, liver mitochondria-PE, total plasma lipids and adipose tissue was low, and 20∶1trans was not detected. Dietarytrans fatty acids had no consistent effects on the overall fatty acid composition of the different tissue lipids. It is conclude thattrans fatty acids from PHFO, HFO and PHSBO have no significant effects on the fatty acid accretion in the fetal piglet.     

The incorporation of orally administered radiolabeled dihomo γ-linolenic acid (20∶3ω6) into rat tissue lipids and its conversion to arachidonic acid

Radioactivity from orally administered radiolabeled dihomo-γ-linolenic acid (20∶3ω6) was recovered from the liver, plasma and brain lipid fractions. After administration the fatty acid was metabolized to arachidonic acid, the 22 carbon chain length fatty acid, and was also β-oxidized. However, 22 hr after administration of [1-¹⁴C]20∶3 between one-third and one-half of the recovered radioactivity was still associated with dihomo-γ-linolenic acid in the liver and plasma lipid fractions. Orally administered dihomo-γ-linolenic acid is incorporated into lipid fractions and is, therefore, available in the metabolic pool for PGE₁ synthesis.     

Plasma lipids are affected similarly by dietary lauric or palmitic acid in gerbils and monkeys

To compare the relative impact of dietary lauric acid (12∶0) and palmitic acid (16∶0) on plasma lipids, two fat-sensitive species, Mongolian gerbils and cebus monkeys, were fed cholesterol-free, purified diets enriched with either 12∶0-rich or 16∶0-rich fats, while all other fatty acids were held constant by selective blending of up to five natural fats or oils. The two gerbil diets (40 en% from fat) allowed for an 8 en% exchange between 12∶0 and 16∶0, and the monkey diets (31 en% from fat) allowed for 6 en% exchange beteen these two fatty acids. Eight gerbils received the diets for eight weeks, and 12 cebus monkeys were fed each diet in a cross-over design for up to 22 wk. Both diets resulted in similar plasma cholesterol, triglyceride, and high density lipoprotein cholesterol concentrations within each species. Additionally, separation of cebus lipoproteins by discontinuous density-gradient ultracentrifugation failed to show any dietary differences in concentration or composition of the three major lipoprotein classes (d<1.019, 1.019–1.055, and 1.055–1.168 g/mL). Thus, in two species sensitive to manipulations in dietary fat while consuming cholesterol-free diets, 16∶0 was not hypercholesterolemic relative to 12∶0. Based on a paper presented at the PORIM International Palm Oil Congress (PIPOC) held in Kuala Lumpur, Malaysia, September 1993.     

Biosynthesis of long-chain polyenoic acids from arachidonic acid in cultures of enriched spermatocytes and spermatids from mouse testis

Primary spermatocytes (PS), round spermatids (RS) and condensing spermatids (CS) from mouse testes were enriched on Sta-Put 1 X g density gradients and cultured for 22 or 44 hr in the presence of [1-14C]arachidonate. Mass and radioactivity were measured by gas radiochromatography of constituent fatty acids of the various complex lipid classes fractionated by thin layer chromatography. Patterns and levels of incorporation were compared with those of whole testis, both in vitro and in vivo. The 20:4, 22:4, 22:5, 24:4 and 24:5 of the germinal cells contained levels of radioactivity in each lipid class which were consistent with an important role for the germinal cells in long-chain polyenoic acid (LCPA) metabolism. Cells which represented later stages of spermatogenesis (RS, CS) incorporated much higher percentages and absolute amounts of radioactivity into the fatty acids derived from 20:4 by elongation-desaturation pathways than did PS or whole testis in vitro. These differences were most pronounced in triacylglycerol of CS. Distributions of mass and radioactivity among lipid classes suggest synthesis of triacylglycerol by CS with a high degree of specificity for 22 or 24 carbon LCPA at the sn-3 position.     

Trans fatty acids. 2. Fatty acid composition of the brain and other organs in the mature female pig

Female pigs were fed from three wk of age and up to two years a diet containing partially hydrogenated fish oil (PHFO, 28%trans monoenoic fatty acids), partially hydrogenated soybean oils (PHSBO, 36%trans fatty acids) or lard. No consistent differences were found between PHFO and PHSBO with regard to incorporation oftrans fatty acids in organ lipids, buttrans incorporations were highly organ-specific. Notrans fatty acids were detected in brain phosphatidylethanolamine (PE). The incorporation of monoenoictrans isomers, as a percentage of totalcis + trans, in other organs was highest in subcutaneous adipose tissue and liver mitochondria PE, followed by blood lipids with the lowest level in heart PE. The percentage oftrans isomers compared with that of dietary lipids was consistently lower for 20∶1, compared with 18∶1 in organs from PHFO-fed pigs. The only effect of dietarytrans fatty acids on the fatty acid pattern of brain PE was an increased level of 22∶5n−6. Heart PE and total serum lipids of pigs fed the hydrogenated fats contained higher levels of 18∶2n−6, and these lipids of the PHFO-fed group also contained slightly elevated amounts of 20∶3n−6, 18∶3n−3 and 20∶5n−3. Liver mitochondria PE of the PHFO group also contained higher levels of 20∶3n−6 and 22∶5n−6. Dietarytrans fatty acids caused a consistent decrease of saturated fatty acids compensated by increased levels of monoenes. Thus, it may be concluded that dietary long-chaintrans fatty acids in PHFO behaved similarly metabolically to 18∶1-trans in PHSBO in pigs, without noticeable influence on brain PE composition and with moderate to slight effects on the fatty acid profile of the other organs.     

Effect of dietary fatty acids on Δ5 desaturase activity and biosynthesis of arachidonic acid in rat liver microsomes

The effect of different fatty acids supplemented to a fat-free diet on the activity of Δ5 desaturase was studied. Fat-free diet produces a reduction in the conversion of eicosa-8,11,14-trienoic acid to arachidonic acid. The addition of thecis-ω6 acids, linoleic, γ-linolenic or arachidonic to the diet produces an increase of eicosatrienoic acid desaturation, shifting Δ5 desaturase activity towards the controls on a balanced diet. This reactivation is apparently produced by induction of enzyme biosynthesis since linoleate effect was suppressed by simultaneous cycloheximide injection. On the contrary, no changes in Δ5 desaturation activity were found when the diet was supplemented with palmitic or 9-trans,12-trans-linoleic acid. The changes on the activity of Δ5 desaturase were compared with the fatty acid composition of plasma and liver microsomes.     

Application of standard addition to eliminate conjugated linoleic acid and other interferences in the determination of total Trans fatty acids in selected food products by infrared spectroscopy

A novel and rapid (5 min) attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopic method AOCS Cd 14d-99 for the determination of total isolated trans fatty afids, which absorb at 966 cm^‐, was recently developed, collaboratively studied, and applied to food products containing 1–50% rans fat (as percentage of total fat). Attempts to apply the ATR-FTIR method to biological matrices of low trans fat and/or low total fat content, and to dairy and other products were not satisfactory due to interfering IR absorptions in the trans region. One group of interfering compounds with absorption bands near 985 and 948 cm⁻¹ was the cis/trans positional isomes of conjugated linoleic acid (CLA) found in dairy and meat products from ruminants at levels of <1% (as percentage of total fat). In the present study, we modified the ATR-FTIR method to overcome matrix interferences. This modification, which consisted of applying the standard addition technique to the ATR-FTIR determination, was also applied to several food products, namely, dairy products, infant formula and salad oil dressing, which successfully eliminated interfering absorbances that impacted on accuracy. The presence of <1% CLA in two butter and two cheese products containing 6.8, 7.5, 8.5, and 10.4% trans fatty acids (as a percentage of total fat) would have led to errors of −11.6, 10.4, 17.6 and 34.6%, respectively, in trans fat measurements had the standard addition technique not been used. The applicability of ATR-FTIR to the quantitation of food products is discussed.     

Distribution oftrans-6-hexadecenoic acid, 7-methyl-7-hexadecenoic acid and common fatty acids in lipids of the ocean sunfishMola mola

Lipids extracted from various tissues of four individual sunfish have been shown to contain thetrans-6-hexadecenoic acid previously reported for marine turtles, a metridium and a jelly fish, and also the 7-methyl-7-hexadecenoic acid recently isolated from one sunfish liver oil sample. The other fatty acids present were qualitatively typical of marine lipids in general. Unusual quantitative details included high percentages of 18∶0 and 20∶4ω6, which are also found in the Atlantic leatherback turtle and presumably linked to a similar diet of jellyfish and to other common factors. In a sample of lipids from intestinal contentstrans-6-hexadecenoic acid was found to be the predominant C₁₆ monoene, and was accompanied by comparatively large amounts of the 7-methyl-7-hexadecenoic acid. This observation and other fatty acid details are compatible with an exogenous origin for these acids and jellyfish, etc., as a predominant dietary material for the ocean sunfish.