Gunhild Hølmer

A new thiobarbituric acid (TBA) method for determining free malondialdehyde (MDA) and hydroperoxides selectively as a measure of lipid peroxidation

A new thiobarbituric acid (TBA) method for selectively determining free malondialdehyde (MDA) and lipid hydroperoxides is described. Partitioning of MDA and hydroperoxides in a Bligh and Dyer extraction was studied with pure substances. It was shown that MDA was present in the methanol-water phase exclusively, and the hydroperoxides (polar as well as nonpolar) were found in the chloroform phase. Therefore, the TBA test on these phases determines MDA directly present in the sample and the MDA formed during a ferric ion-catalyzed cleavage of hydroperoxides, respectively. The MDA present in the methanol-water phase was not bound to amino groups. Hydroperoxide values obtained with the present method corresponded well with a colorimetric determination of peroxides and showed better linearity for higher amounts of hydroperoxides. The possibility of using an iron chelator for preventing hydroperoxide cleavage during the TBA reaction, making the determination selective for MDA without Bligh and Dyer extraction, was investigated. The iron chelator did not completely inhibit peroxide cleavage; therefore, it is necessary to perform the extraction before the TBA determination. The method is suitable for samples with low fat content such as cod mince.

Visual acuity and erythrocyte docosahexaenoic acid status in breast-fed and formula-fed term infants during the first four months of life

It has been recognized that preterm infants have a more rapid development of visual acuity if fed human milk or a formula enriched with the long-chain polyunsaturated fatty acid (LCPUFA) docosahexaenoic acid (DHA) compared to a standard formula devoid of LCPUFA. Few studies have addressed whether the same is also true in term infants. The aim of the present study was to follow visual acuity and fatty acid composition in red blood cells (RBC) for the first 4 mon of life in 17 breast-fed and 16 formula-fed term infants. The formula used did not contain LCPUFA, but contained 1.7 wt% α-linolenic acid, and the linoleic/α-linolenic acid ratio was 8.5. The increase in visual acuity measured by Teller acuity cards developed more rapidly in breast-fed infants compared to formula-fed infants (P<0.001). This was parallelled by a decrease in DHA of RBC in formulafed infants, and with a significantly lower level at two and four months as compared to breast-fed infants. The content of DHA in milk from the breast-feeding mothers was high compared to other Western countries. The difference in visual acuity between the two feeding groups could be due to differences in DHA status as reflected by the RBC levels, but other explanations are possible. Intervention studies are required to verify if development of visual acuity in term formula-fed infants is dependent on the DHA level of formula.

Regiospecific analysis of triacylglycerols using allyl magnesium bromide

A method for the regiospecific analysis of triacylglycerols (TAG), using the Grignard reagent allyl magnesium bromide (AMB) to partially deacylate TAG, is described. 1,3-Distearoyl-2-oleoyl-glycerol (SOS) and 1,3-didecanoyl-2-palmitoyl-glycerol (CPC) were reacted with AMB. From the resulting mixture, the four different classes of partial acylglycerols and TAG were isolated, and the mole ratios between stearic acid and oleic acid, or decanoic acid and palmitic acid, respectively, were determined in each fraction. Different approaches of calculating the composition of the fatty acids in positionssn-1(3) andsn-2 of the original TAG were compared. For thesn-2 position, the best estimate was the direct determination of the fatty acid composition of 2-monoacylglycerol (MAG). Mole percentages of stearic acid and decanoic acid in thesn-1(sn-3) positions of SOS and CPC, respectively, were most accurately estimated from the fatty acid compositions of TAG and 2-MAG according to the formula: 1.5×TAG−0.5×2-MAG. Using AMB and the present method of calculation, the results obtained were more accurate and showed smaller standard derivations than those obtained using other common deacylating agents, such as ethyl magnesium bromide or pancreatic lipase.

Effect of Formula Supplemented with Docosahexaenoic Acid andγ-linolenic Acid on Fatty Acid Status and Visual Acuity in Term Infants

BACKGROUND Docosahexaenoic acid is present in high concentration in retina and does not influence visual development in preterm infants. It is still under discussion whether docosahexaenoic acid is important for visual development in term infants. METHODS Thirty-seven infants fed formula for a median of 14 days were randomized at median age of 25 days to three formulas: a) DHAGF: 0.3 wt% docosahexaenoic acid and 0.5 wt% gamma-linolenic acid; b) DHAF 0.3 wt% docosahexaenoic acid; or c) STF: standard formula without long-chain polyunsaturated fatty acids and 17 breast-fed infants were observed, using blood samples and anthropometric measurements from 1 to 4 months of age. At 4 months, visual acuity was measured by swept steady-state visual evoked potential. A cross-sectional study on 25 breast-fed infants was carried out as a reference group for the analyses. RESULTS Infants fed the two docosahexaenoic acid-supplemented formula had relative docosahexaenoic acid concentrations in red blood cell phospholipids almost as high as those in breast-fed infants, whereas infants in the standard formula group had significantly lower levels. The addition of gamma-linolenic acid to the formula had a positive effect on red blood cell arachidonic acid levels, compared with levels obtained using fish oil only. Visual acuity was significantly different among all feeding groups (analysis of variance; p = 0.05, means +/- standard deviation: breast-fed, 0.37+/-0.06 logMAR; DHAF and DHAGF combined, 0.40+/-0.07 logMAR; and standard formula 0.44+/-0.07 logMAR. However, there was no statistical difference among the formula groups. In a multiple regression analysis including all formula-fed infants, weight at delivery (p = 0.002), but not type of formula, was significantly associated with visual acuity at 4 months of age. CONCLUSIONS The addition of docosahexaenoic acid resulted in concentrations in red blood cells at similar levels as those in breast-fed infants, whereas the increase in visual acuity did not reach significance. The addition of gamma-linolenic acid resulted in higher arachidonic acid concentrations in red blood cells.

Effects of dietary linseed oil and marine oil on lipid peroxidation in monkey liver in vivo and in vitro

Diets rich in linoleic acid (CO) from corn oil, or in linoleic acid and either α-linolenic acid (LO) based on linseed oil or n−3 fatty acids (MO) from menhaden oil were fed to male and female Cynomolgus monkeys for 15 wk. In the liver a 40% reduction of α-tocopherol occurred in the MO group relative to the CO and LO groups followed by increased formation of lipofuscinin vivo. A four-fold increase of α-tocopherol in the MO diet (MO+E) brought the level in the liver to that found with CO and LO. The increased peroxidation in the MO group in the liver phospholipids was associated with the replacement of 60% of the n−6 fatty acids by n−3 fatty acids from menhaden oil. Similar fatty acid profiles were found in groups fed MO and MO+E, respectively. Compared to the CO fed group, feeding α-linolenic acid only resulted in a slight incorporation of n−3 fatty acids in the liver membranes mainly due to a direct incorporation of α-linolenic acid. However, in monkeys fed menhaden oil more than 30% of the total fatty acids in the liver phospholipids were n−3 fatty acids. The various diets did not influence the activity of liver catalase (EC 1.11.1.6) nor superoxide dismutase (EC 1.15.1.1), but glutathione-peroxidase activity (EC 1.11.1.9) was higher in monkeys fed the MO diet. The catalase activity in females was 20% higher than in males. In anin vitro assay, liver microsomes from monkeys fed the MO diet or the MO diet supplemented with tocopherol produced similar amounts of thiobarbituric acid reactive substances and at a much higher rate than microsomes from the CO and LO groups. It appeared that α-tocopherol did not protect long-chain n−3 C20 and C22 fatty acids as well as n−6 fattya acids against peroxidation. The present data showed that monkeys were not fully able to compensate for increased peroxidative stress but a four-fold supplement of vitamin E to the diets reduced the oxidation.

Coenzyme Q10 in the diet-daily intake and relative bioavailability

The coenzyme Q10 content of the average Danish diet was estimated from consumption data and from analysis of food items to be 3-5 mg coenzyme Q10 per day, primarily derived (64% of the total) from meat and poultry. To investigate if coenzyme Q10 was absorbed to any significant degree from a food item, a randomized cross-over study with single doses of coenzyme Q10 (30 mg/person), administered either as a meal or as capsules, was carried out in healthy subjects. The serum coenzyme Q10 concentration increased significantly, and the maximum concentrations did not differ significantly for the two forms of administration. The study indicates that coenzyme Q10 is present in food items and absorbed to a significant degree. Thus, dietary coenzyme Q10 may contribute to the plasma coenzyme Q10 concentration.

Incorporation ofcis- andtrans-octadecenoic acids into the membranes of rat liver mitochondria

The incorporation of dietary isomeric fatty acids into the membranes of liver mitochondria was investigated. Three groups of rats were fed diets containing 3% sunflower seed oil plus 15%, 20%, or 25% partially hydrogenated arachis oil. A fourth group was fed 25% partially hydrogenated arachis oil, but no sunflower seed oil. All diets were given for 3, 6, or 10 weeks. After 10 weeks, the content oftrans fatty acids in the lipids of the mitochondrial membranes was 15–19% of the total fatty acids. The composition of thetrans- and thecis-octadecenoic acids in the lipids of the mitochondrial membranes was similar for all groups supplemented with sunflower seed oil (SO), irrespective of time and dietary level of partially hydrogenated arachis oil (HAO). Thecis 18∶1 (n−8), which was a major isomer of the partially hydrogenated arachis oil, was almost excluded from the mitochondrial fatty acids. Likewise, the content oftrans 18∶1 (n−8) was considerably lower in the mitochondrial lipids than in the diet. On the contrary, the content oftrans 18∶1 (n−6) was higher in the mitochondrial lipids than in the diet. In the group fed without sunflower seed oil, isomers of linoleic acid and arachidonic acid were observed in the lipids of mitochondrial membranes.

Effect of modified dairy fat on postprandial and fasting plasma lipids and lipoproteins in healthy young men

Fatty acid profile of milk fat can be modified by cow feeding strategies. Our aim was postprandially and after 4 wk to compare the effect of a modified milk fat (M diet) [with 16% of the cholesterolemic saturated fatty acid (C12–16) replaced by mainly oleic and stearic acids] with the effect of D diet, including a conventional Danish milk fat on plasma lipids and lipoproteins. A side effect of the cow feeding regime was a 5% (w/w) increase in trans fatty acid in M diet. Eighteen subjects were fed for two periods of 4 wk strictly controlled isoenergetic test diets with 40% of energy from total fat and the same content of dietary cholesterol in a randomized study with cross-over design. Contrary to expectations, fasting low density lipoprotein (LDL) cholesterol concentration did not differ after the experimental periods. However, M diet resulted in a higher fasting total triacylglycerol concentration compared to D diet (P=0.009). Postprandial samples were taken at two different occasions (i) at day 21, after breakfast and lunch and (ii) on the last day of the study 2, 4, 6, and 8 h after a fat load. Postprandial plasma triacylglycerol and chylomicron triacylglycerol showed higher peak values after D diet than M diet (interaction effect, diet × times P<0.05). In conclusion, M diet did not lower LDL cholesterol compared to D diet. Thus any cholesterol-lowering effect of oleic and stearic acids may have been obscured by the high content of cholesterol-raising saturated fatty acids in milk fat. A higher content of the trans fatty acids in M diet might have counteracted the cholesterol neutral/decreasing effect and increased plasma triacylglycerol.

Changes in lipid composition of germinating barley embryo

Barley seeds,Hordeum vulgare, var. Kenia, were dissected before and after 5 days of germination, to distinguish between the scutellum, the coleoptile half of the embryo and the coleorhiza half of the embryo. Total lipids were extracted from each fraction and analyzed by thin layer chromatography and gas liquid chromatography. In tissues from the coleoptile and coleorhiza halves of the embryo there was a concurrent disappearance of triglycerides with a marked increase of esterified sterols and esterified sterol glucosides. In the scutellum there was also a change in triglycerides, but the variations in contents of esterified sterols and esterified sterol glucosides were much smaller. Mono- and digalactolipids were virtually absent from embryonic tissue. The amounts of linoleic and linolenic acids in esterified sterol glucosides were increased after 5 days of germination in all the embryonic tissues, especially in the coleoptile half. In sterol esters, linoleic acid comprised nearly half of the total fatty acids, and the desaturation after 5 days of germination was much less pronounced.

Separation and detection of phospholipid hydroperoxides in the low nanomolar range by a high performance liquid chromatography/irothiocyanate assay

A new method for the detection of phospholipid hydroperoxides in the low nanomolar range has been developed by using a high performance liquid chromatography system combined with a post-column reaction. The detection is based on the oxidation of Fe(II) to Fe(III) by hydroperoxides and the subsequent formation of an ironthiocyanate complex, which can be determined spectrophotometrically at 505 nm. By this method it is possible to separate and quantitate phosphatidylethanolamine hydroperoxide and phosphatidylcholine hydroperoxide formed in erythrocyte ghost membranes during photo-oxidation.