Partially Hydrogenated Fish Oil Research Articles

Silver ion solid‐phase extraction chromatography for the analysis of trans fatty acids

Summary This article describes the application of silver ion solid-phase extraction (Ag + -SPE) to the separation of the fatty acid methyl esters (FAMEs)prepared from a partially hydrogenated fish oil (PHFO) with a trans fatty acid (FA) content of 41%. The complex FAME mixture wasresolved into the following FAME fractions: saturated FA, trans-monounsaturated FA (trans-MUFA), cis-MUFA, and several polyunsatu-rated FA (PUFA) fractions with mixed geometric and positional isomers. Fractions were analyzed by gas chromatography (GC) using a 100 mcyanopropyl polysiloxane (CPS) coated capillary column. The GC analysis of the Ag + -SPE fractions allowed for the identification of the trans-andcis-MUFAandotherFAMEpreparedfromPHFO. Introduction As early as 1966 Morris [1] had concluded that argentation chroma-tography was third in importance to GC and thin-layer chromato-graphy (TLC) for the analysis of lipids. This optimistic predictionwas largely based on separations achieved by using Ag + -TLC toresolve FAMEs with different numbers and geometric configura-tions of double bonds. These results were essential to complementGC separations of FAMEs on packed columns that provided reso-lution determined by chain length and number of double bonds, butcould not resolve geometric isomers. One might have thought thatthe subsequent development of the very long, highly polar GC ca-pillary columns that now make it possible to resolve most geo-metric and positional isomers and PUFAs would have phased outthe need for silver ion (Ag

Effects of partially‐hydrogenated fish oil on lipid and lipoprotein metabolism in guinea pigs

This study was designed to examine the effects of partially hydrogenated fish oil (PHFO), compared with fish oil (FO) used as a control, on serum lipids, lipoprotein profile and aortic lesion development in guinea pigs fed an atherogenic diet. Adult male Hartley guinea pigs (n=20; 10 per group) were fed a diet supplemented with 8.5 % PHFO or FO for 12 weeks. Diets contained 0.25 % cholesterol (C). The total trans fatty acid content of the PHFO diet was 4 %. PHFO‐fed animals had a significantly increased serum concentrations of triacylglycerol, total cholesterol, LDL‐C, and sdLDL‐C (P<0.001) compared with the FO group. In contrast, serum HDL‐C concentrations did not differ between the groups. Analysis of the ascending aorta and aortic arch revealed no lesion development in both treatment groups but PHFO‐fed guinea pigs showed a distinct lipid accumulation in the aortic arch when compared to the FO group. Results from this study demonstrate that guinea pigs provide a good model for studying the effects of PHFO on the lipid/lipoprotein metabolism. However, no signs of atherosclerosis in the aorta (lesion development) could be established under the used experimental conditions.

Acute postprandial effect of hydrogenated fish oil, palm oil and lard on plasma cholesterol, triacylglycerol and non-esterified fatty acid metabolism in normocholesterolaemic males

The majority of research has focused on the association between trans unsaturated fatty acids (TUFA) from hydrogenated vegetable oils and heart disease even though TUFA are also produced from hydrogenated fish oil. We compared the acute effect of three solid fats on postprandial cholesterol, triacylglycerol (TAG) and NEFA concentrations in normocholesterolaemic males. Eight healthy male volunteers consumed each of the three 40 g fat meals (partially hydrogenated fish oil (PHFO), palm oil and lard) in random order and blood samples were drawn at 2, 4, 6 and 8 h thereafter for lipid analysis. The postprandial response in plasma TAG, TAG-rich lipoprotein-TAG (TRL-TAG), total cholesterol and plasma NEFA, measured as the area under the postprandial curve, was not significantly different between the three meals (P>0.05), which varied in MUFA, PUFA and TUFA content. There was no marked elevation of longer-chain fatty acids (C20-22, cis or trans isomers) into the TRL-TAG fraction following the PHFO meal even though they provided 40 % of the total fatty acids in the PHFO meal. The postprandial TRL-TAG response to PHFO was expected to be higher, as it is higher in TUFA, lower in PUFA and similar in saturated fatty acid composition compared with the lard and palm oil test meals. The absence of a higher postprandial response following ingestion of PHFO could be as a result of reduced absorption and increased oxidation of long-chain fatty acids (both cis and trans isomers).

Absorption of very-long-chain saturated fatty acids in totally hydrogenated fish oil

Partially hydrogenated fish oil (PHFO) contains a high amount of trans fatty acids (TFA). Total hydrogenation results in a minimal amount of TFA, but a high content of very-long-chain saturated fatty acids (VLCSFA). Absorption and metabolism of VLCSFA from totally hydrogenated fish oil (THFO) were studied in rats. Groups of eight rats were fed one of four diets containing 40 g soyabean oil (SBO)/kg (low-fat diet), 150 g SBO/kg (SBO diet), 40 g PHFO/kg (PHFO diet) or 40 g THFO/kg (THFO diet) for 4 weeks. A lower absorption coefficient of the fat content was found in the THFO group (61 %) compared with the other groups (PHFO 95 %, SBO 99 %, low fat 98 %; which was mainly due to reduced absorption of VLCSFA. A reduced weight gain was found for the THFO group compared with the other groups, but this was only significant when compared with the SBO group Faecal fat excretion (dry weight) was markedly increased in the THFO group (47 %), which was 2.4, 4.8 and 8.3 times higher compared with the groups fed PHFO, SBO and low-fat diets respectively. Serum total cholesterol was reduced for the PHFO and THFO groups whereas serum triacylglycerol was increased for the PHFO group compared with the other groups Animals fed THFO diet had an increased content of 20:0 and 22:0 in the serum triacylglycerol fraction whereas only 20:0 was increased in the serum phospholipid fraction The low absorption coefficient of THFO must be considered if this fat is to be used for consumption by animals or man.

Effect on plasma lipids and lipoproteins of replacing partially hydrogenated fish oil with vegetable fat in margarine

We have compared the effects on lipoproteins and haemostatic variables of two hard margarines with similar functional properties, one traditional margarine containing partially hydrogenated fish oil (PHFO), and one experimental margarine based on vegetable oil (VO). Both were all-purpose cooking margarines with nearly identical functional properties. Trans fatty acids from PHFO in the traditional margarine were replaced mostly by saturated, monounsaturated and trans fatty acids of vegetable origin in the new formulation. Both test margarines contained approximately the same amount of cis polyunsaturated fatty acids. Sixteen female normolipidaemic students consumed each diet with the two test margarines for 14 d in random order (crossover design). The amount of fat was 31% energy in the PHFO diet and 32% energy in the VO diet. The test margarines provided approximately 26% energy in both diets. In the PHFO diet 7.8% of the energy was derived from trans fatty acids and 9.2% from saturated fatty acids (12:0, 14:0 and 16:0) while in the VO diet, 1.1% energy was derived from trans fatty acids and 13.3% from saturated fatty acids (12:0, 14:0 and 16:0). The natural content of cholesterol in PHFO was deliberately not balanced by addition of cholesterol to the VO diet, thus the PHFO diet contained 215 mg and the VO diet 86 mg cholesterol per 8.5 MJ. LDL-cholesterol concentration was 19% higher in subjects on the PHFO diet compared with the VO diet (P < 0.01). The ratio LDL-cholesterol:HDL-cholesterol was 12.6% higher in subjects on the PHFO diet compared with the VO diet (P < 0.01). The level of apolipoprotein (apo)A-I was 6% lower in subjects on the PHFO diet compared with the VO diet (P < 0.01). The ratio apoB:apoA-I was 10.4% higher in subjects on the PHFO diet than on the VO diet (P < 0.01). There were no significant differences in total cholesterol, HDL-cholesterol, triacylglycerols, apoB, lipoprotein(a) and haemostatic variables between the diets. Our results demonstrate that PHFO, with its unfavourable effects on plasma lipids, can be replaced by vegetable oils in margarine without appreciable loss of functional properties but with significant improvement in the effects on plasma lipoproteins.

Studies of dihydroxyacetone phosphate acyltransferase in rat small intestine. Subcellular localization and effect of partially hydrogenated fish oil and clofibrate.

The subcellular localization of dihydroxyacetone phosphate acyltransferase (DHAPAT) activity in rat small intestine was investigated by Nycodenz-gradient centrifugation. We found that DHAPAT had a predominant peroxisomal distribution, with a separate enzyme activity located in the microsomal fraction, the same distribution as found in rat liver. The effect of feeding rats on a diet with 20% (w/w) partially hydrogenated fish oil (PHFO) or 0.3% clofibrate on the activity of DHAPAT in rat small intestine and liver was studied. Both 20% PHFO and 0.3% clofibrate gave a 1.8-fold stimulation of the specific activities of DHAPAT in peroxisomes of the small intestine, whereas in the liver 20% PHFO gave a 1.4-fold stimulation and 0.3% clofibrate a 1.6-fold stimulation of the total DHAPAT activities in the postnuclear supernatant. The specific activities of DHAPAT in liver were not affected.

Trans fatty acids. 4. Effects on fatty acid composition of colostrum and milk

trans Isometric fatty acids of partially hydrogenated fish oil (PHFO) consist of trans 20:1 and trans 22:1 in addition to the trans isomers of 18:1, which are abundant in hydrogenated vegetable oils, such as in partially hydrogenated soybean oil (PHSBO). The effects of dietary trans fatty acids in PHFO and PHSBO on the fatty acid composition of milk were studied at 0 (colostrum) and 21 days postpartum in sows. The dietary fats were PHFO (28% trans), or PHSBO (36% trans) and lard. Sunflower seed oil (4%) was added to each diet. The fats were fed from three weeks of age throughout the lactation period of Experiment 1. In Experiment 2 PHFO or "fully" hydrogenated fish oil (HFO) (19% trans), in comparison with coconut oil (CF) (0% trans), was fed with two levels of dietary linoleic acid, 1 and 2.7%, from conception throughout the lactation period. Feeding trans-containing fats led to secretion of trans fatty acids in the milk lipids. Levels of trans 18:1 and trans 20:1 in milk lipids, as percentages of total cis + trans 18:1 and cis + trans 20:1, respectively, were about 60% of that of the dietary fats, with no significant differences between PHFO and PHSBO. The levels were similar for colostrum and milk. Feeding HFO gave relatively less trans 18:1 and trans 20:1 fatty acids in milk lipids than did PHFO and PHSBO. Only low levels of cis + trans 22:1 were found in milk lipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Chain Elongation of Fatty Acids in Rat Small Intestine: Subcellular Localization and Effects of Clofibrate and Partially Hydrogenated Fish Oil

Clofibrate treatment was found to increase by a factor of two the activity of malonyl-CoA-dependent palmitic acid chain elongation in crude 'microsomal' fractions isolated from rat small intestine. A slight stimulation was indicated also after feeding a high fat diet containing partially hydrogenated fish oil (PHFO). This effect was, however, not statistically significant. Subcellular localization of this chain elongation activity was studied by Nycodenz gradient centrifugation. Chain elongation of palmitic acid was observed only in fractions containing high esterase activity, suggesting a microsomal localization. Similar distributions were seen after feeding the peroxisomal proliferators clofibrate and PHFO. Chain-shortening activity was found in the fractions containing high catalase activity, confirming our earlier studies suggesting a peroxisomal localization of this activity also in rat small intestine.

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

The effects of dietary trans 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 of trans fatty acids into brain PE (phosphatidylethanolamine) were non-detectable or very low (less than 0.1%). The incorporation of 18:1 trans into heart-PE, liver mitochondria-PE, total plasma lipids and adipose tissue was low, and 20:1 trans was not detected. Dietary trans fatty acids had no consistent effects on the overall fatty acid composition of the different tissue lipids. It is concluded that trans fatty acids from PHFO, HFO and PHSBO have no significant effects on the fatty acid accretion in the fetal piglet.

Trans fatty acids. 1. Growth, fertility, organ weights and nerve histology and conduction velocity in sows and offspring

Effects of dietary trans fatty acids on the pre- and postnatal growth and development in pigs were studied with special emphasis on nervous tissue. In experiment 1, female pigs were fed partially hydrogenated fish oil (PHFO) (28% trans) or soybean oil (PHSBO) (36% trans), in comparison with lard (0% trans) from weaning (3 wk) through the first reproduction cycle (up to 2 yr). In experiment 2, female pigs were fed two fish oils (33 and 19% trans) in comparison with coconut oil (0% trans) in diets with low and high levels of linoleic acid (18:2n-6 cis, cis) from gestation until their offspring were three wk old. Compared with the trans-free fats, the trans-containing fats had no effect on growth and development, feed consumption and utilization or on the weight of the brain, heart, kidneys, liver, lungs or spleen in the adult sows and their offspring. No effects from the experimental fats were found on histology and conduction velocity of the peroneal nerve. An increased number of the sows fed PHFO had fertility problems compared with those fed lard and PHSBO in Expt. 1, but no similar effects were seen in Expt. 2. It is concluded that consumption of trans fatty acids with 18-22 carbon atoms from PHFO and with 18 carbon atoms from PHSBO at levels that were 5 to 12 times higher than those normally consumed by humans had no detrimental effects on female pigs or their offspring during pregnancy and lactation.

Rapid stimulation of liver palmitoyl-CoA synthetase, carnitine palmitoyltransferase and glycerophosphate acyltransferase compared to peroxisomal β-oxidation and palmitoyl-CoA hydrolase in rats fed high-fat diets

Key enzymes involved in oxidation and esterification of long-chain fatty acids were investigated in male rats fed different types and amounts of oil in their diet. A diet with 20% (w/w) fish oil, partially hydrogenated fish oil (PHFO) and partially hydrogenated soybean oil (PHSO) was shown to stimulate the mitochondrial and microsomal palmitoyl-CoA synthetase activity (EC 6.2.1.3) compared to soybean oil-fed animals after 1 week of feeding. Rapeseed oil had no effect. Partially hydrogenated oils in the diet resulted in significantly higher levels of mitochondrial glycerophosphate acyltransferase compared to unhydrogenated oils in the diet. Rats fed 20% (w/w) rapeseed oil had a decreased activity of this mitochondrial enzyme, whereas the microsomal glycerophosphate acyltransferase activity was stimulated to a comparable extent with 20% (w/w) rapeseed oil, fish oil or PHFO in the diet. Increasing the amount of PHFO (from 5 to 25% (w/w)) in the diet for 3 days led to increased mitochondrial and microsomal palmitoyl-CoA synthetase and microsomal glycerophosphate acyltransferase activities with 5% of this oil in the diet. The mitochondrial glycerophosphate acyltransferase was only marginally affected by increasing the oil dose. Administration of 20% (w/w) PHFO increased rapidly the mitochondrial and microsomal palmitoyl-CoA synthetase, carnitine palmitoyltransferase and microsomal glycerophosphate acyltransferase activities almost to their maximum value within 36 h. In contrast, the glycerophosphate acyltransferase and palmitoyl-CoA hydrolase (EC 3.1.2.2) activities of the mitochondrial fraction and the peroxisomal β-oxidation reached their maximum activities after administration of the dietary oil for 6.5 days. This sequence of enzyme changes (a) is in accordance with the proposal that an increased cellular level of long-chain acyl-CoA species act as metabolic messages for induction of peroxisomal β-oxidation and palmitoyl-CoA hydrolase i.e., these enzymes are regulated by a substrate-induced mechanism, and (b) indicates that, with PHFO, a greater part of the activated fatty acids are directed from triacylglycerol esterification and hydrolysis towards oxidation in the mitochondria. It is also conceivable that the mitochondrial β-oxidation is proceeding before the enhancement of peroxisomal β-oxidation.

Effect of a high-fat diet with partially hydrogenated fish oil on long-chain fatty acid metabolizing enzymes in subcellular fractions of rat liver

Hepatic metabolism of long-chain fatty acids were studied in young male rats fed a semisynthetic diet containing 20% ( w w ) partially hydrogenated fish oil (PHFO) 2, with or without 2% ( w w ) linoleic acid. The enzymic activities involved in the formation and breakdown of long-chain acyl-CoA were both increased in the animals fed the semisynthetic diet, compared to pellet-fed control animals. Thus, the specific palmitoyl-CoA synthetase activity increased slightly in both the mitochondrial (1.4-fold) and the microsomal (1.6-fold) fractions. In the peroxisome-enriched fraction the activity was increased (about 2.6-fold) only on addition of linoleic acid to the diet. The data are consistent with an increased catabolism of long-chain fatty acids by a peroxisomal and a mitochondrial pathway. Thus, the total carnitine palmitoyltransferase activity increased 2-fold in the mitochondrial fraction, and was partly prevented by added linoleic acid. Peroxisomal β-oxidation activity was also increased (about 7-fold) in livers of PHFO-fed rats, but did not change when linoleic acid was added. The PHFO-fed rats also revealed elevated capacity for hydrolysis of palmitoyl-CoA in both the mitochondrial (2.4-fold) and the cytosolic (2.0-fold) fractions and the latter was almost completely and selectively prevented by added linoleic acid. The s̄ values of mitochondria and peroxisomes varied with the dietary regime, and some of the observed changes in the specific activities of the fatty acid metabolizing enzymes with multiple subcellular localization can be explained as an effect of changes in the s̄ values of the organelles. Thus, the s̄ value of mitochondria increased 1.8-fold as a result of PHFO feeding, but was fully prevented by linoleic acid in the diet. On the other hand, the s̄ values of peroxisomes decreased by about 50% on feeding a PHFO diet, and by about 25% with added linoleic acid.

The effect of feeding fish oils, vegetable oils and clofibrate on the ketogenesis from long chain fatty acids in hepatocytes.

Groups of rats were fed diets containing 25% fish oil (FO), 25% soybean oil, 25% partially hydrogenated fish oil (PHFO), 25% partially hydrogenated soybean oil (PHSO), 25% partially hydrogenated coconut oil or 0.3% clofibrate for 3 wk. After the animals were fasted for 24 hr, hepatocytes were isolated and ketogenesis from added palmitate, linoleate cis and trans, arachidonate and docosahexaenoate was measured. Ketogenesis after oil feeding was significantly stimulated (two- to threefold) only in cells from the PHFO- and PHSO-fed rats. The stimulation was most apparent with the long chain unsaturated fatty acids as substrates. These fatty acids were relatively poor ketone body precursors in control hepatocytes. Essential fatty acid deficiency did not seem to be the reason for this stimulation. Clofibrate also stimulated ketogenesis significantly (1.5- to 3-fold). The degree of stimulation increased with chain length and degree of unsaturation of the substrate. The activity of the enzyme 2,4-dienoyl-CoA reductase was also studied in the same groups. Its activity was stimulated about fourfold in the clofibrate-treated rats and to a lesser extent by the PHFO, PHSO and FO diets. The activity showed no correlation with the content of unsaturated fatty acids in the diet or their oxidation in isolated hepatocytes. The 2,4-dienoyl-CoA reductase, therefore, does not seem to be a regulatory enzyme in the metabolism of dietary polyunsaturated fatty acids. It is concluded that an induction of the peroxisomal beta-oxidation system most likely is involved in the reported increases in ketogenesis from very long chain polyunsaturated fatty acids.

Long-term effects of high-fat diets on peroxisomal beta-oxidation in male and female rats.

In weanling male rats a 4-fold increase of heart triacylglycerols was observed after three days on a high-fat diet containing partially hydrogenated fish oil (PHFO). In female rats this increase was only about 50%. No significant differences were observed between female and male rats in the fatty acid composition of the accumulated lipids. The initial level of peroxisomal beta-oxidation activity was similar in male and female rats in both liver and heart. After three weeks of receiving high-fat diets, the rats showed a marked increase in peroxisomal beta-oxidation activity with PHFO in the diet and less with soybean oil (SO), confirming previous studies with male rats. Catalase activity was similarly affected in hearts of both sexes. In male rats the levels of peroxisomal beta-oxidation observed after three weeks of feeding on the high-fat diets were found to be maintained, both in liver and heart, during a feeding period of three months. The response to high-fat diets in females, however, seems to be further accentuated after three months of feeding, resulting in a capacity of peroxisomal beta-oxidation in liver of about three times that of the male rats when calculated on a total body-weight basis.

Chain-length specificities of mitochondrial and peroxisimal β-oxidation of fatty acids in livers of rainbow trout ( Salmo gairdneri)

1. 1. Peroxisomes and mitochondria were prepared from livers of rainbow trout fed diets containing either 15% crude fish oil (CFO) or 11.5% partially hydrogenated fish oil (PHFO) plus 3.5% CFO. 2. 2. Peroxisomal preparations from the two dietary groups showed similar rates and substrate specificity patterns for acyl-CoA oxidation. 3. 3. The peroxisomal oxidation rate was highest with 12:0-CoA and decreased with increasing chain length, being negligible with 22-carbon acyl-CoA's. The trans isomer of 18:1( n-9) was oxidized at a higher rate than the cis isomer only by peroxisomes from the PHFO + CFO group. 4. 4. Mitochondria prepared from both groups of fish exhibited a broad chain-length specificity for the oxidation of acylcarnitines. Both polyunsaturated and trans monoenoic fatty acids were readily oxidized.

Lipid metabolism in rainbow trout ( Salmo gairdnerii) fed diets containing partially hydrogenated fish oil

1. 1. Rainbow trout were fed diets containing either 15% crude fish oil (CFO), 11.5% partially hydrogenated fish oil (PHFO) and 3.5% CFO, or 15% PHFO. 2. 2. Fish maintained on PHFO exhibited the highest death and lowest growth rates. CFO- and PHFO + CFO-fed fish had similar death and growth rates. After 15 months, PHFO + CFO fish had larger hearts and livers than fish fed CFO. 3. 3. Plasma cholesterol and triglyceride concentrations were similar in CFO and PHFO + CFO-fed fish and lowest in the group fed PHFO. 4. 4. Tissue lipids of PHFO + CFO fish were richer in 18:1 and less unsaturated than those of the CFO group. PHFO was richer in 22:1 than CFO, but the 22:1 contents of tissue lipids of PHFO + CFO and CFO fish were similar and less than those of the dietary oils. 5. 5. Cyanide-insensitive palmitoyl-CoA oxidation activity in liver was highest in trout fed the PHFO + CFO diet, while carnitine palmitoyltransferase was not affected by diet. 6. 6. It is suggested that the excess of long chain monoenes in relation to polyunsaturates in a PHFO + CFO diet causes an induction of the peroxisomal β-oxidation of fatty acids in fish liver.

Comparison of lipid status in the hearts of piglets and rats on short term feeding of marine oils and rapeseed oils

A series of 4 experiments with piglets and one experiment with rats has been conducted to establish the cardiac lipid status of weanling (3 weeks old) male animals fed fats with different contents of docosenoic fatty acids. Experimental fats were rapeseed oil (RSO) (48.0% 22:1), refined fish oil (RFO) (14.6% 22:1), partially hydrogenated fish oil (PHFO) (14.3% 22:1) and lard (0% 22:1) combined with sunflower seed oil (SFO) in different proportions in diets with 21% total fat. Lipidosis could not be detected in piglets as increased heart weights, by chemical assay for myocardial contents of triglycerides, or by accumulation of docosenoic fatty acids or nonesterified fatty acids (NEFA). In rats, diets with RSO at a level of 16% increased myocardial triglyceride and docosenoic fatty acid contents about 7 times while the effect on cardiac NEFA was inconsistent. Histological examinations of the hearts revealed stainable intracellular fat droplets in some piglets fed 16% RSO for 8 to 13 days, but not after 2, 4 and 6 and 16, 19 and 22 days of feeding. After 10 days of feeding, mild to moderate histological lipidosis was found in piglets fed diets containing 2% or more of 22:1 fatty acids, with no significant difference between RSO, RFO and PHFO in this respect. The same diets in rats gave about 5 times more histological lipidosis than in piglets. This is attributed to a difference in species response, the rat reacting in a more pronounced manner than the piglet. The cardiac lipidosis no-effect level in piglets corresponded to a daily intake of docosenoic fatty acids of 0.4 g per kg body weight. Mild lipidosis was also found in a few animals on docosenoic acid-free diets.