Effects of an isocaloric healthy Nordic diet on insulin sensitivity, lipid profile and inflammation markers in metabolic syndrome -- a randomized study (SYSDIET).

BACKGROUND: Different healthy food patterns may modify cardiometabolic risk. We investigated the effects of an isocaloric healthy Nordic diet on insulin sensitivity, lipid profile, blood pressure and inflammatory markers in people with metabolic syndrome. METHODS: We conducted a randomized dietary study lasting for 18-24 weeks in individuals with features of metabolic syndrome (mean age 55 years, BMI 31.6 kg m(-2) , 67% women). Altogether 309 individuals were screened, 200 started the intervention after 4-week run-in period, and 96 (proportion of dropouts 7.9%) and 70 individuals (dropouts 27%) completed the study, in the Healthy diet and Control diet groups, respectively. Healthy diet included whole-grain products, berries, fruits and vegetables, rapeseed oil, three fish meals per week and low-fat dairy products. An average Nordic diet served as a Control diet. Compliance was monitored by repeated 4-day food diaries and fatty acid composition of serum phospholipids. RESULTS: Body weight remained stable, and no significant changes were observed in insulin sensitivity or blood pressure. Significant changes between the groups were found in non-HDL cholesterol (-0.18, mmol L(-1) 95% CI -0.35; -0.01, P = 0.04), LDL to HDL cholesterol (-0.15, -0.28; -0.00, P = 0.046) and apolipoprotein B to apolipoprotein A1 ratios (-0.04, -0.07; -0.00, P = 0.025) favouring the Healthy diet. IL-1 Ra increased during the Control diet (difference -84, -133; -37 ng L(-1) , P = 0.00053). Intakes of saturated fats (E%, beta estimate 4.28, 0.02; 8.53, P = 0.049) and magnesium (mg, -0.23, -0.41; -0.05, P = 0.012) were associated with IL-1 Ra. CONCLUSIONS: Healthy Nordic diet improved lipid profile and had a beneficial effect on low-grade inflammation.

The antioxidant capacity of milk--the application of different methods in vitro and in vivo.

Milk contains a wide array of compounds with established or putative pro- or anti-oxidant function. The functions of these compounds have been intensively studied. This review focusses on some important aspects in this wide field namely the methodology for measurement of the total antioxidant capacity (TAC), the content of TAC and some related compounds in human and animal milks and infant formulas, and the effect of milk intake on antioxidant status in the body and on the activity of dietary flavonoids as studied in vitro and in vivo. Regarding methodology TAC in milk can be measured by spectrophotometric and electrochemical methods and some of their characteristics are reviewed. Milk, whey, high-molecular-weight and low-molecular-weight (LMW) fractions of whey have all been found to have antioxidant capacity using these techniques. The major antioxidant in the LMW fraction has been identified as urate. An extensive literature survey was made regarding data on the antioxidant capacity and related variables of milk obtained from different sources (human milk, infant formulas and animal milk) and subjected to different treatments. Differences in TAC between milks from different sources have been observed but due to the variety of techniques used no clear pattern is evident at present. Another important aspect is the putative effects of the intake of milk products on the antioxidant status of the consumer. A few studies performed in adults and premature infants are reviewed and it is stated that too little information is available to make any firm conclusions in this regard. Finally, a high interest has been devoted to the possible interference of milk with the antioxidant properties of flavonoid-rich food like tea. Most in vitro studies show an inhibition by milk on tea flavonoid activity whereas the results from the corresponding in vivo studies are equivocal. Our general conclusion is that several compounds in various milk fractions contribute to the antioxidant capacity of milk and that much further work is needed to unravel the complex interactions among the pro- and antioxidants, and their putative health effects on the consumer.

Occurrence of selenoprotein enzyme activities and mRNA in bovine mammary tissue.

To elucidate the possible role of selenoproteins for milk formation and mammary gland physiology, the activities of selenoprotein enzymes and the expression of selenoprotein genes were studied in the bovine mammary gland. Messenger RNA was demonstrated for selenoprotein P, thioredoxin reductase 1, and for glutathione peroxidase (GPx) 1, 3, and 4. Significant differences in mRNA expression between the cows were seen for GPx 1 and GPx 3. The enzyme activity of glutathione peroxidase varied approximately 16-fold among cows, and the activity of thioredoxin reductase and the concentration of soluble Se varied approximately 6-fold among cows. There were positive correlations between glutathione peroxidase activity, thioredoxin reductase activity, and soluble Se, the correlation between glutathione peroxidase activity and soluble Se being the strongest. Furthermore, selenoprotein P expression correlated with GPx 1 mRNA expression and with soluble Se. There was also a correlation between glutathione peroxidase activity and the mRNA expression of GPx 1. The general conclusion from the data was that the activity of glutathione peroxidase and thioredoxin reductase and the mRNA expression of selenoprotein P and GPx 1 and 3 were influenced by Se status, but the expression of GPx 4 and thioredoxin reductase 1 were not. These results indicate that the Se status in mammary tissue is an important regulator of selenoprotein activity and expression, but that other factors are also in operation.

Effect of sphingosine and other amphiphilic amines on the biosynthesis of phosphatidylethanolamine and other glycerolipids in isolated rat hepatocytes.

The importance of ethanolamine and sphingosine as precursors of phosphoethanolamine was investigated by incubating them with [3H]glycerol and isolated rat hepatocytes. Sphingosine (0.1--0.5 mM) stimulated the synthesis of phosphatidylethanolamine from [3H]glycerol, but the stimulation by ethanolamine was more pronounced. Furthermore, more phosphoethanolamine accumulated in the heptatocytes after incubation with ethanolamine than after incubation with sphingosine. It is concluded that ethanolamine is the most important phosphoethanolamine precursor in rat liver. Higher concentrations of sphingosine caused accumulation of [3H]phosphatidate and inhibition of total glycerolipid synthesis in isolated hepatocytes, when incubated in the presence of [3H]glycerol. These effects were very similar to those of fenfluramine and norfenfluramine described previously. Simpler cationic amphiphilic amines, like oleoylamine and octadecyltrimethylammonium bromide, also caused these effects. Variation of alkyl chain length and amphiphile charge showed that both a positive charge and a certain alkyl chain length were necessary for interference with phosphatidate metabolism. A much wider range of compounds inhibited total glycerolipid synthesis from [3H]glycerol.

Structural requirements of the phospholipid substrate for phospholipid N-methylation in rat liver.

The role of endogenous phospholipid substrates for phospholipid methylation was investigated in rat liver microsomes. The amount of phosphatidylethanolamine could be drastically reduced by treatment of microsomes with an amino group-blocking compound, methylacetimidate. Simultaneously, the formation of labelled phospholipids from S-adenosyl[Me-3H]methionine decreased, indicating that the amount of endogenous substrate influenced the reaction rate. Phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine and phosphatidylmonoethylethanolamine added as dispersions to untreated or treated microsomes stimulated phospholipid methylation, whereas several other phospholipids were inactive. In other experiments the role of phospholipid substrates in intact cells was studied. Cultured rat hepatocytes were enriched in different phospholipids by preincubation with different amino alcohols, and the effects of phospholipid methylation was measured by incubation with [Me-14C]methionine. Phospholipid methylation was significantly stimulated after preincubation with ethanolamine, monomethylethanolamine, monoethylethanolamine and 2-aminobutanol. The results show that both the number and chain length of N-alkyl substituents on phosphatidylethanolamine, as well as other changes in the ethanolamine moiety, will affect the ability of different phospholipids to act as methyl acceptors.

Phospholipid degradation in isolated rat hepatocytes. Metabolism of intracellularly formed dilinoleoyl-dipalmitoyl- and dimyristoylglycerophosphocholine.

The aim of this work is to describe the role of different phospholipases in hepatic phospholipid catabolism. Therefore isolated rat hepatocytes enriched in labeled dilinoleoyl-, dipalmitoyl- or dimyristoylglycerophosphocholine were prepared by pulse incubation with [3H]glycerol and 14C-labeled fatty acid. The labeled cells were chased up to 4 h in a tracer-free medium and the degradation of different phosphatidylcholines studied. After a 2-h chase about 40% of dilinoleoyl-, 70% of dipalmitoyl- and 30% of dimyristoylglycerophosphocholine were degraded. From the positional distribution of 14C-labeled fatty acid and the change in the doubly labeled molecular species of phospholipids, it was concluded that tb degradation of dilinoleoylglycerophosphocholine and that of phosphatidylethanolamine could be accounted for by the action of phospholipase A1, while the degradation of dipalmitoylglycerophosphocholine proceeded through the action of phospholipase A2. Dimyristoylglycerophosphocholine was probably cleaved by the combined action of both phospholipases A1 and A2. Up to 10 mM tetracain, added to the chase medium, effectively blocked the action of both phospholipase activities. A considerable part of 2-linoleoyl- and 1-palmitoylglycerophosphocholine liberated during the chase was reutilized for phosphatidylcholine synthesis without further degradation.

Metabolism of different monoacylphospholipids in isolated hepatocytes and the intact rat.

The 1-[3H] palmitoyl, 2-[3H] oleoyl, and 2-[14C] linoleoyl derivatives of sn-glycero-3-phosphoethanolamine and the corresponding derivatives of sn-glycero-3-phosphocholine were injected intraportally to rats and their incorporation into liver lipids was studied 15 min thereafter. Both the uptake by the liver and the degree of acylation was higher for the unsaturated compounds. The uptake of lysophosphatidylethanolamine was higher than that of lysophosphatidlycholine. The metabolism of 1-lysophosphatidylethanolamine was also studied in isolated hepatocytes. The degree of hydrolysis was much more prominent than in vivo. After injecting 2-[14C] linoleoyl derivatives, a large part of the 14C was recovered in the dienoic phospholipids. Subfractionation by reversed-phase partition chromatography showed that the isotope was located in the palmitoyllinoleoyl and stearoyl-linoleoyl fraction. The 100 X stearoly/(palmitoyl + stearoyl) ratio was 84 in dienoic phosphatidylethanolamine and 59 in dienoic phosphatidylcholine. This preference for stearic acid is significantly larger than in other pathways yielding dienoic phospholipids. It can be concluded that the monoacylphospholipid acyltransferase reactions operating at positions 1 or 2 yield different saturated acyl chain profiles in phosphatidylethanolamine and phosphatidylcholine of a specific unsaturation. This may be important in the regulation of the fatty acid composition of the membrane phospholipids.

Effects of human pancreatic lipase-colipase and carboxyl ester lipase on eicosapentaenoic and arachidonic acid ester bonds of triacylglycerols rich in fish oil fatty acids.

Fish oil chylomicrons, obtained from mesenteric duct chyle of rats fed [3H]20:5 and [14C]20:4 or [3H]20:5 and [14C]18:2 in a fish oil emulsion, were incubated with human pancreatic lipase-colipase, human carboxyl ester lipase (CEL) and human duodenal contents. With duodenal contents, the triacylglycerols labelled with [3H]20:5 and [14C]20:4 were rapidly converted to free fatty acids (FFA) and monoacylglycerols. Also during incubation with lipase-colipase the [3H]- and [14C]triacylglycerols disappeared completely and at equal rates, but in this case much [3H]20:5 and [14C]20:4 accumulated in diacylglycerols. When CEL was also added, the rate of disappearance of [3H]- and [14C]triacylglycerols increased and the radioactivity of diacylglycerols decreased markedly. During incubation of chylomicrons labelled with [3H]20:5 and [14C]18:2 with lipase-colipase, the rates of hydrolysis of [3H]- and [14C]triacylglycerols were similar, but more [3H]20:5 than [14C]18:2 accumulated in diacylglycerols. The accumulation of [3H]diacylglycerol was reduced by adding CEL. Also when fatty acids were analyzed by gas chromatography, 20:5 was enriched in remaining triacylglycerol and in diacylglycerol after incubation with lipase-colipase alone. The data thus indicate that both lipase-colipase and CEL participate in the hydrolysis of 20:5 and 20:4 ester bonds of dietary triacylglycerol.

Purification of selenoprotein P from human plasma.

Selenoprotein P was partially purified (> 1000-fold) from human plasma in four chromatographic steps using 75Se-labeled selenoprotein P secreted by HepG2 cells in culture as a marker. The purified preparation was injected into mice and monoclonal antibodies, which precipitated the labeled protein, were generated. Neither of two different monoclonal antibodies had cross-reactivity with plasma from five animal species. Antibodies were coupled to agarose, and selenoprotein P was purified from human plasma by immunoaffinity chromatography followed by chromatography on heparin agarose. With two different matrix-bound monoclonal antibodies, the purification procedure gave two bands on SDS-PAGE with mobilities corresponding to 61 and 55 kDa. Both bands stained for carbohydrate and showed increased electrophoretic mobility after enzymatic deglycosylation. Immunoaffinity chromatography removed approx. one-third of the selenium from plasma or 0.4 mumol Se/l at a total selenium concentration of 1.1 mumol/l, indicating that selenoprotein P constituted this proportion of total plasma selenium in healthy US blood donors.

Hydrolysis of chylomicron polyenoic fatty acid esters with lipoprotein lipase and hepatic lipase.

The lipolysis of rat chylomicron polyenoic fatty acid esters with bovine milk lipoprotein lipase and human hepatic lipase was examined in vitro. Chylomicrons obtained after feeding fish oil or soy bean oil emulsions were used as substrates. The lipolysis was followed by gas chromatography or by using chylomicrons containing radioactive fatty acids. Lipoprotein lipase hydrolyzed eicosapentaenoic (20:5) and arachidonic acid (20:4) esters at a slower rate than the C14-C18 acid esters. More 20:5 and 20:4 thus accumulated in remaining tri- and diacylglycerols. Eicosatrienoic, docosatrienoic and docosahexanoic acids exhibited an intermediate lipolysis pattern. When added together with lipoprotein lipase, hepatic lipase increased the rate of lipolysis of 20:5 and 20:4 esters of both tri- and diacylglycerols. Addition of NaCl (final concentration 1 M) during the course of lipolysis inhibited lipoprotein lipase as well as the enhancing effect of hepatic lipase on triacylglycerol lipolysis. Hepatic lipase however, hydrolyzed diacylglycerol that had already been formed. Chylomicron 20:4 and 20:5 esters thus exhibit a relative resistance to lipoprotein lipase. It is suggested that the tri- and diacylglycerol species containing these fatty acids may accumulate at the surface of the remnant particles and act as substrate for hepatic lipase during a concerted action of this enzyme and lipoprotein lipase.