Acute elevations of medium-and long-chain fatty acids have different impacts on endothelium-dependent vasodilation in humans

It has previously been shown that acute elevation of long-chain fatty acids (LCFA) impairs endothelium-dependent vasodilation (EDV) in humans. In this study, we tested the hypothesis that an elevation of both medium-chain fatty acids (MCFA) and LCFA affects the endothelium differently from LCFA elevation alone. Ten healthy volunteers received an intravenous infusion of Structolipid^≿ (structured TG, MCFA/LCFA ratio 1∶1) and heparin for 2 h, while another 10 subjects received an infusion of Intralipid^≿ (LCFA only) and heparin. EDV and endothelium-independent vasodilation (EIDV) were studied in the forearm after local administration of methacholine chloride (2 and 4 μg/min) and sodium nitroprusside (5 and 10 μg/min). Forearm blood flow was determined by venous occlusion plethysmography. Intralipid and heparin increased circulating FA levels from 0.2±0.1 to 1.4±0.5 mmol/L (P<0.001) and reduced EDV by 20% (P<0.01). Although Structolipid and heparin increased circulating FA levels to a similar extent (from 0.4±0.1 to 1.8±0.4 mmol/L after 2 h), EDV was not significantly changed. EIDV increased slightly during both interventions (P<0.05). In conclusion, an acute elevation of LCFA attenuated EDV, whereas an elevation of both MCFA and LCFA did not influence EDV. Thus, FA composition seems to be of importance for EDV in healthy humans.     

Changes in Plasma LDL and HDL Composition in Patients Undergoing Cardiac Surgery

Changes of lipoprotein composition have been mainly reported in conditions of sepsis. This study characterized compositional changes in LDL and HDL during the acute phase response following cardiac surgery with cardiopulmonary bypass. Twenty-one patients undergoing cardiac surgery were included in this study. Blood samples were drawn before operation and on day 2 post-surgery. In parallel to plasma lipids and antioxidant status, lipoproteins were analyzed for lipid, apolipoprotein (apo), hydroperoxide and alpha-tocopherol content. Beyond decreases in lipid concentrations and antioxidant defenses, cardiac surgery induced substantial modifications in plasma lipoproteins. ApoB decrease in LDL fraction (−46%; P < 0.0001) reflected a marked reduction in the circulating particle number. LDL cholesteryl ester content relative to apoB concentration remained unchanged post-surgery while triglyceride (+113%; P < 0.001), free cholesterol (+22%; P < 0.05) and phospholipid (+23%; P < 0.025) were raised relative to apoB indicating increased particle size. In HDL, an abrupt rise of apoSAA (P < 0.05) was observed together with a decrease of apoA1 (−22%; P < 0.005). Cholesteryl ester content in HDL fraction decreased in parallel to apoA1 concentration while triglycerides, free cholesterol and phospholipids increased relative to apoA1. In contrast to unchanged alpha-tocopherol content, hydroperoxide content was increased in LDL and HDL. By comparison to sepsis, cardiac surgery induces a comparable reduction in circulating LDL but a more limited decrease in HDL particles. Furthermore, in contrast, cardiac surgery induces an increase in polar and non-polar lipids, as well as of particle size in both LDL and HDL. M. Hacquebard is recipient of a fellowship from the Danone Institute, Belgium.     

Rosuvastatin Enhances the Catabolism of LDL apoB‐100 in Subjects with Combined Hyperlipidemia in a Dose Dependent Manner

Dose‐associated effects of rosuvastatin on the metabolism of apolipoprotein (apo) B‐100 in triacylglycerol rich lipoprotein (TRL, d < 1.019 g/ml) and low density lipoprotein (LDL) and of apoA‐I in high density lipoprotein (HDL) were assessed in subjects with combined hyperlipidemia. Our primary hypothesis was that maximal dose rosuvastatin would decrease the apoB‐100 production rate (PR), as well as increase apoB‐100 fractional catabolic rate (FCR). Eight subjects received placebo, rosuvastatin 5 mg/day, and rosuvastatin 40 mg/day for 8 weeks each in sequential order. The kinetics of apoB‐100 in TRL and LDL and apoA‐I in HDL were determined at the end of each phase using stable isotope methodology, gas chromatography‐mass spectrometry, and multicompartmental modeling. Rosuvastatin at 5 and 40 mg/day decreased LDL cholesterol by 44 and 54 % (both P < 0.0001), triacylglycerol by 14 % (ns) and 35 % (P < 0.01), apoB by 30 and 36 % (both P < 0.0001), respectively, and had no significant effects on HDL cholesterol or apoA‐I levels. Significant decreases in plasma markers of cholesterol synthesis and increases in cholesterol absorption markers were observed. Rosuvastatin 5 and 40 mg/day increased TRL apoB‐100 FCR by 36 and 46 % (both ns) and LDL apoB‐100 by 63 and 102 % (both P < 0.05), respectively. HDL apoA‐I PR increased with low dose rosuvastatin (12 %, P < 0.05) but not with maximal dose rosuvastatin. Neither rosuvastatin dose altered apoB‐100 PR or HDL apoA‐I FCR. Our data indicate that maximal dose rosuvastatin treatment in subjects with combined hyperlipidemia resulted in significant increases in the catabolism of LDL apoB‐100, with no significant effects on apoB‐100 production or HDL apoA‐I kinetics.     

Effect of liposome-encapsulated hemoglobin on triglyceride, total cholesterol, low-density lipoprotein, and high-density lipoprotein cholesterol measurements

The present study investigated the effect of liposome-encapsulated hemoglobin (LEH), an experimental oxygen-carrying resuscitation fluid, on triglyceride, total cholesterol, and low density lipoprotein (LDL), and high density lipoprotein (HDL) cholesterol measurements. In vivo, the intravenous infusion of LEH (5.6 mL/kg, n=6) elevated serum triglycerides (+92% vs. baseline, P<.05), total cholesterol (+25% vs. baseline, P<.01), LDL cholesterol (+72% vs. baseline, P<.01) and had no effect on serum HDL cholesterol. In addition, LEH did not alter the elevation in serum triglycerides (+302% vs. baseline, P<.01) and LDL cholesterol (+86% vs. baseline, P<.01) induced by lipopolysaccharide (3.6 mg/kg, i.v., n=6). Ex vivo, measurements of triglycerides and total cholesterol as well as LDL and HDL cholesterol in whole blood from naive rats were not changed by the addition of LEH (0–50%, n=6). In vitro, the addition of a fixed concentration of LEH (50%, n=6) to varying concentrations of cholesterol solution (0–50%), or vice versa, had no effect on cholesterol determination. It is therefore concluded that LEH only minimally affects serum levels of triglycerides, total cholesterol, LDL cholesterol, and HDL cholesterol and does not interfere with their measurement.     

Studies on the transfer of tocopherol between lipoproteins

The net transfer of labeled α-tocopherol from donor to acceptor lipoproteins at physiological concentrations was investigated. Labeled lipoproteins were isolated i) followingin vitro addition of [3,4-³H]all rac-α-tocopherol to plasma, or ii) from plasma obtained 12–16 h after ingestion by normal subjects of an oral dose (100 mg each) of 2R,4′R,8′R-α-[5,7-(C²H₃)₂]tocopheryl acetate and 2S,4′R′,R-α-[5-C²H₃]tocopheryl acetate. A constant amount (on a protein basis) of labeled lipoprotein was incubated with an increasing amount of unlabeled acceptor lipoprotein for 2 h at 37°C. No discrimination between stereoisomers of α-tocopherol was detected. Labeled VLDL and labeled LDL (very low and low density lipoproteins, respectively) tended to retain their labeled tocopherol. Labeled high density lipoproteins (HDL) readily transferred the labeled tocopherol to VLDL (>60% transferred), while the transfer to LDL was dependent upon the ratio of labeled HDL/LDL with a lower net transfer at higher ratios. This dependency of the distribution of tocopherol upon the ratio of HDL/LDL was also observedin vivo. The tocopherol/mg HDL protein was measured in 11 subjects with varying HDL levels. As the %HDL in the plasma increased from 14 to 50%, the tocopherol/HDL protein also increased (r²=0.37,P<0.05).     

Electronegative Low-Density Lipoprotein is Associated with Dense Low-Density Lipoprotein in Subjects with Different Levels of Cardiovascular Risk

Dyslipidemias and physicochemical changes in low-density lipoprotein (LDL) are very important factors for the development of coronary artery disease (CAD). However, pathophysiological properties of electronegative low-density lipoprotein [LDL(−)] remain a controversial issue. Our objective was to investigate LDL(−) content in LDL and its subfractions (phenotypes A and B) of subjects with different cardiovascular risk. Seventy-three subjects were randomized into three groups: normolipidemic (N; n = 30) and hypercholesterolemic (HC; n = 33) subjects and patients with CAD (n = 10). After fasting, blood samples were collected and total, dense and light LDL were isolated. LDL(−) content in total LDL and its subfractions was determined by ELISA. LDL(−) content in total LDL was lower in the N group as compared to the HC (P < 0.001) and CAD (P = 0.006) groups. In the total sample and in those of the N, HC, and CAD groups, LDL(−) content in dense LDL was higher than in light LDL (P = 0.001, 0.001, 0.001, and 0.033, respectively) The impact of LDL(−) on cardiovascular risk was reinforced when LDL(−) content in LDL showed itself to have a positive association with total cholesterol (β = 0.003; P < 0.001), LDL-C (β = 0.003; p < 0.001), and non-HDL-C (β = 0.003; P < 0.001) and a negative association with HDL-C (β = −0.32; P = 0.04). Therefore, LDL(−) is an important biomarker that showed association with the lipid profile and the level of cardiovascular risk.     

Effect of dietary cholesterol on low density lipoprotein-receptor, 3-hydroxy-3-methylglutaryl-CoA reductase, and low density lipoprotein receptor-related protein mRNA expression in healthy humans

We investigated the possibility that dietary cholesterol downregulates the expression of low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase genes of circulating mononuclear cells in vivo in healthy humans. We also studied the variations of the LDL receptor-related protein (LRP) gene in the same conditions. Dieters (n=5) were submitted to a 4-d fat restriction (mean cholesterol intake: 6±4 mg/d), followed by a 7-d cholesterol (a mean of 791±150 mg/d) supplementation. Controls (n=3) did not change their diet. During fat restriction, serum total and LDL cholesterol decreased significantly (P<0.05), and LDL receptor and HMG-CoA reductase mRNA copy numbers in mononuclear cells increased by 57 and 147%, respectively (P<0.05). After reintroducing cholesterol, serum cholesterol was stable whereas LDL receptor and HMG-CoA reductase mRNA decreased by 46 and 72% (P<0.05) and LRP mRNA increased by 59% (P<0.005). The changes in LDL receptor and HMG-CoA reductase mRNA abundance were correlated (r=+0.79, P=0.02) during cholesterol reintroduction as were LDL receptor and LRP mRNA levels, but negatively (r=−0.70, P=0.05). Also, 70% of the variability in LRP mRNA (P<0.005) was explained by dietary cholesterol. Thus, the basic mechanisms regulating cellular cholesterol content, the coordinate feedback repression of genes governing the synthesis and uptake of cholesterol, are operating in vivo in humans. However, serum cholesterol did not increase in response to dietary cholesterol, suggesting that these mechanisms may not play as predominant a role as previously believed in the short-term control of serum cholesterol in vivo in humans. A new finding is that LRP gene is also sensitive to dietary cholesterol, suggesting that it may participate in the control of serum cholesterol. Further in vivo studies in humans are warranted to explore the molecular mechanisms of the physiological response to dietary cholesterol in humans.     

Dietary linoleic acid-induced hypercholesterolemia and accumulation of very light HDL in steers

This experiment was designed to study the effects in fattening steers of n−6 PUFA supplementation on the plasma distribution and chemical composition of major lipoproteins (TG-rich lipoproteins: d<1.006 g/mL; intermediate density lipoproteins + LDL: 1.019<d<1.060 g/mL; light HDL: 1.060<d<1.091 g/mL; and heavy HDL: 1.091<d<1.180 g/mL). For a period of 70 d, animals [454±20 d; 528±36 kg (mean±SD)] were given a control diet (diet C, n=6) consisting of hay and concentrate mixture (54 and 46% of diet dry matter, respectively) or the same diet supplemented with sunflower oil (4% of dry matter), given either as crushed seeds (diet S, n=6) or as free oil continuously infused into the duodenum through a chronic canula to avoid ruminal PUFA hydrogenation (diet O, n=6). Plasma lipids increased in steers given diet S (×1.4, P<0.05) and diet O (×2.3, P<0.05), leading to hyperphospholipemia and hypercholesterolemia. With diet S, hypercholesterolemia was associated with higher levels of light (×1.4, P<0.05) and heavy HDL (×1.3, NS). With diet O, it was linked to higher levels of light HDL (×1.8, P<0.005) and to very light HDL accumulation within density limits of 1.019 to 1.060 g/mL, as demonstrated by the apolipoprotein A-I profile. Diet O favored incorporation of 18∶2n−6 into polar (×2.2, P<0.05) and neutral lipids (×1.5 to ×8, P<0.05) at the expense of SFA, MUFA, and n−3 PUFA. Thus, protection of dietary PUFA against ruminal hydrogenation allowed them to accumulate in plasma lipoproteins, but the effects of hypercholesterolemia on animal health linked to very light HDL accumulation remain to be elucidated.     

Response of plasma lipids to dietary cholesterol and wine polyphenols in rats fed polyunsaturated fat diets

This experiment was designed to evaluate the effects of dietary red wine phenolic compounds (WP) and cholesterol on lipid oxidation and transport in rats. For 5 wk, weanling rats were fed polyunsaturated fat diets (n−6/n−3=6.4) supplemented or not supplemented with either 3 g/kg diet of cholesterol, 5 g/kg diet of WP, or both. The concentrations of triacylglycerols (TAG, P<0.01) and cholesterol (P<0.0002) were reduced in fasting plasma of rats fed cholesterol despite the cholesterol enrichment of very low density lipoprotein + low density lipoprotein (VLDL+LDL). The response was due to the much lower plasma concentration of high density lipoprotein (HDL) (−35%, P<0.0001). In contrast, TAG and cholesteryl ester (CE) accumulated in liver (+120 and +450%, respectively, P<0.0001). However, the cholesterol content of liver microsomes was not affected. Dietary cholesterol altered the distribution of fatty acids mainly by reducing the ratio of arachidonic acid to linoleic acid (P<0.0001) in plasma VLDL+LDL (−35%) and HDL (−42%) and in liver TAG (−42%), CE (−78%), and phospholipids (−28%). Dietary WP had little or no effect on these variables. On the other hand, dietary cholesterol lowered the α-tocopherol concentration in VLDL+LDL (−40%, P<0.003) and in microsomes (−60%, P<0.0001). In contrast, dietary WP increased the concentration in microsomes (+21%, P<0.0001), but had no effect on the concentration in VLDL+LDL. Cholesterol feeding decreased (P<0.006) whereas WP feeding increased (P<0.0001) the resistance of VLDL+LDL to copper-induced oxidation. The production of conjugated dienes after 25 h of oxidation ranged between 650 (WP without cholesterol) and 2,560 (cholesterol without WP) μmol/g VLDL+LDL protein. These findings show that dietary WP were absorbed at sufficient levels to contribute to the protection of polyunsaturated fatty acids in plasma and membranes. They could also reduce the consumption of α-tocopherol and endogenous antioxidants. The responses suggest that, in humans, these substances may be beneficial by reducing the deleterious effects of a dietary overload of cholesterol.     

Long-chain polyunsaturated fatty acids in plasma lipids of obese children

Fatty acid composition of plasma phospholipids (PL), triglycerides (TG), and sterol esters (STE) was determined by high-resolution capillary gas-liquid chromatography in 22 obese children (age: 13.7±1.4 y, body weight relative to normal weight for height: 170±24%, mean ±SD) and compared with data obtained in 25 age-matched healthy controls. There were no differences in the levels of linoleic acid (LA, C_18∶2n-6) in any of the plasma fractions from the obese children and the controls. Obese children exhibited significantly higher values of arachidonic acid (AA, C_20∶4n-6) than controls both in PL (12.6 [2.4] vs. 8.3 [1.4], % wt/wt, [median (interquartile range)],P<0.001) and STE (7.3 [1.8] vs. 6.0 [1.1],P<0.05). Similarly, obese children showed higher values than controls for dihomo-γ-linolenic acid (DHGLA, C_20∶3n-6) in PL (4.0 [0.5] vs. 3.0 [0.6],P<0.001), TG (0.4 [0.1] vs. 0.2 [0.1],P<0.001), and STE (0.9 [0.1] vs. 0.7 [0.1],P<0.01), and for γ-linolenic acid (C_18∶3n-6) in STE (1.1 [0.2] vs. 0.8 [0.2],P<0.001). The AA/LA ratios were higher in obese children than in controls in PL (0.68 [0.16] vs. 0.42 [0.09],P<0.0005) and STE (0.16 [0.04] vs. 0.12 [0.02],P<0.05), whereas the AA/DHGLA ratios were lower in TG of obese children than in controls (3.40 [0.64] vs. 5.10 [1.75],P<0.005). Plasma glucose concentrations were inversely related to AA in TG (r=0.53,P<0.05), and plasma TG concentrations were inversely related to AA in PL and STE (r=−0.49,P<0.05 andr=−0.48,P<0.05) and to the AA/DHGLA ratios in PL (r=−0.57,P<0.01),TG (r=−0.56,P<0.01) and STE (r=−0.56,P<0.01). We conclude that the significantly higher values of n-6 long-chain polyunsaturated fatty acids (LCP) in plasma lipids of obese children than in age-matched controls may be caused by an enhanced activity of Δ6-desaturation, and we speculate that elevated fasting immunoreactive insulin seen in obese children (19.4±8.0 μU/mL) may stimulate synthesis of n-6 LCP fatty acids.     

Dose-Dependent Effects of Docosahexaenoic Acid Supplementation on Blood Lipids in Statin-Treated Hyperlipidaemic Subjects

The objective of the study was to evaluate potential benefits of docosahexaenoic acid (DHA) rich fish oil supplementation as an adjunct to statin therapy for hyperlipidaemia. A total of 45 hyperlipidaemic patients on stable statin therapy with persistent elevation of plasma triglycerides (averaging 2.2 mmol/L) were randomised to take 4 g/day (n = 15) or 8 g/day (n = 15) of tuna oil or olive oil (placebo, n = 15) for 6 months. Plasma lipids, blood pressure and arterial compliance were assessed initially and after 3 and 6 months in 40 subjects who completed the trial. Plasma triglycerides were reduced 27% by 8 g/day DHA-rich fish oil (P < 0.05) but not by 4 g/day when compared with the placebo and this reduction was achieved by 3 months and was sustained at 6 months. Even though total cholesterol was already well controlled by the statin treatment (mean initial level 4.5 mmol/L), there was a further dose-dependent reduction with fish oil supplementation (r = −0.344, P < 0.05). The extent of total cholesterol reduction correlated (r = −0.44) with the initial total cholesterol levels (P < 0.005). In the subset with initial plasma cholesterol above 3.8 mmol/L, plasma very low density lipoprotein (VLDL), intermediate-density lipoprotein (IDL) and low-density lipoprotein (LDL) were isolated and assayed for cholesterol and apolipoprotein B (apoB) at the commencement of the trial and at 3 months of intervention. Fish oil tended to lower cholesterol and apoB in VLDL and raise both in LDL. There were no changes in IDL cholesterol, IDL apoB and high-density lipoprotein cholesterol. The results demonstrate that DHA-rich fish oil supplementation (2.16 g DHA/day) can improve plasma lipids in a dose-dependent manner in patients taking statins and these changes were achieved by 3 months. Fish oil in addition to statin therapy may be preferable to drug combinations for the treatment of combined hyperlipidaemia.     

Fatty acid composition of muscle fat and enzymes of storage lipid synthesis in whole muscle from beef cattle

Enhanced intramuscular fat content (i.e., marbling) in beef is a desirable trait, which can result in increased product value. This study was undertaken with the aim of revealing biochemical factors associated with the marbling trait in beef cattle. Samples of longissimus lumborum (LL) and pars costalis diaphragmatis (PCD) were taken from a group of intact crossbred males and females at slaughter, lipids extracted, and the resulting FAME examined for relationships with marbling fat deposition. For LL, significant associations were found between degree of marbling and myristic (14∶0, r=0.55, P<0.01), palmitic (16∶0, r=0.80 P<0.001), stearic (18∶0, r=−0.58, P<0.01), and oleic (18∶1c-9, r=0.79, P<0.001) acids. For PCD, significant relationships were found between marbling and palmitic (r=0.71, P<0.001) and oleic (r=0.74, P<0.001) acids. Microsomal fractions prepared from PCD muscle were assayed for diacylglycerol acyltransferase (DGAT), lysophosphatidic acid acyltransferase (LPAAT), and phosphatidic acid phosphatase-1 (PAP-1) activity, and the results examined for relationships with degree of intramuscular fat deposition. None of the enzyme activities from PCD displayed an association with marbling fat content, but DGAT specific activity showed significant positive associations with LPAAT (r=0.54, P<0.01), total PAP (r=0.66, P<0.001), and PAP-1 (r=0.63, P<0.01) specific activities. The results on FA compositions of whole muscle tissues provide insight into possible enzyme action associated with the production of specific FA. The increased proportion of oleic acid associated with enhanced lipid content of whole muscle is noteworthy given the known health benefits of this FA.     

Cardiac and vascular structure and function are related to lipid peroxidation and metabolism

The present study investigated possible relationships between left ventricular mass, intima-media thickness of the carotid artery (IMT), total arterial compliance, and lipid status in a population sample of 58 apparently healthy subjects aged 20 to 69. By stepwise multiple regression analysis, including age, blood pressure, and smoking, left ventricular mass index, measured by M-mode echocardiography, increased by 13.0 g/m² for each 1 standard deviation (SD=0.11 μM, r=0.60, P<0.01) increase in plasma malondialdehyde and 9.50 g/m² per SD increase in plasma 8-iso-prostaglandin F_2α in women only (SD=8.88 ng/L, r=0.44, P=0.01). Each 1-SD (SD=0.27 g/L) increase in apolipoprotein B was associated with a 63 μm increase in IMT (r=0.47, P=0.01) and a 0.27 mL/min/m²/mm Hg (r=−0.60, P<0.01) decrease in stroke index/pulse pressure ratio, reflecting total arterial compliance in women. In men, each 1-SD increase in the proportion of stearic acid (18∶0) in serum cholesterol esters (SD=0.12 percent units) reduced the transmitral E/A ratio, measured by Doppler echocardiography, reflecting left ventricular diastolic function, by 0.10 units (r=−0.29, P<0.05). Thus, important cardiovascular characteristics, such as left ventricular mass, left ventricular diastolic function, carotid IMT, and total arterial compliance, were independently predicted by indices of lipid metabolism and peroxidation in apparently healthy subjects.     

Soluble fiber and soybean protein reduce atherosclerotic lesions in guinea pigs. Sex and hormonal status determine lesion extension

These studies were undertaken to assess guinea pigs as potential models for early atherosclerosis development. For that purpose, male, female, and ovariectomized (to mimic menopause) guinea pigs were fed a control or a TEST diet for 12 wk. Differences between diets were the type of protein (60% casein/40% soybean vs. 100% soybean) and the type of fiber (12.5% cellulose vs. 2.5% cellulose/5% pectin/5% psyllium) for control and TEST diets, respectively. Diet had no effect on plasma cholesterol or triacylglycerol (TAG) concentrations; however, there were significant effects related to sex/hormonal status. Ovariectomized guinea pigs had higher plasma cholesterol and TAG concentrations than males or females (P<0.01). In contrast to effects on plasma lipids, hepatic cholesterol and TAG were 50% lower in the TEST groups (P<0.01) compared to controls. Low density lipoproteins (LDL) from guinea pigs fed the TEST diet had a lower number of cholesteryl ester (CE) molecules and a smaller diameter than LDL from controls. Atherosclerotic lesions were modulated by both diet (P<0.0001) and sex (P<0.0001). Guinea pigs fed the TEST diet had 25% less lesion extension whereas males had 20% larger occlusion of the arteries compared to both female and ovariectomized guinea pigs. Significant positive correlations were found between LDL CE and atherosclerotic lesions (r=0.495, P<0.05) and LDL size and fatty streak area (r=0.56, P<0.01). In addition, females fed the TEST diet had the lowest plasma and hepatic cholesterol concentrations, the smallest LDL particles, and the least atherosclerosis involvement compared to the other groups. These data indicate that dietary factors and sex/hormonal status play a role in determining plasma lipids and atherosclerosis in guinea pigs.     

Measurement of apolipoprotein B in various cell lines: Correlation between intracellular levels and rates of secretion

We have standardized simple but sensitive enzyme-linked immunoassays to understand a relationship between intracellular levels and secretion rates of apoB. The assays were based on commercially available antibodies and were specific to human apoB. A monoclonal antibody, 1D1, was immobilized on microtiter wells and incubated with different amounts of low density lipoproteins to obtain a standard curve. Conditioned media were added to other wells in parallel, and the amount of apoB was quantitated from a linear regression curve. To standardize conditions for the measurement of intracellular apoB, cells were homogenized and solubilized with different concentrations of taurocholate. We found that 0.5% taurocholate was sufficient to solubilize all the apoB in HepG2, Caco-2, and McA-RH7777 cells. Next, a standard curve was prepared in the presence of taurocholate and used to determine intracellular levels of apoB in different cell lines. The intracellular levels (pmol/mg cell protein) and the rates of secretion (pmol/mg/h) of apoB100 were positively correlated (r ²=0.81, P=0.0009) in HepG2 cells. Furthermore, a positive correlation (r ²=0.88, P<0.0001) was found between intracellular and secreted apoB42 in stably transfected McA-RH7777 cells. In contrast, no correlation was observed for human apoB28 and apoB18 in stably transfected cells that were secreted either partially associated or completely unassociated with lipoproteins. These studies indicated that the rate of secretion of lipid-associated apoB, but not the lipid-free apoB, was tightly controlled.     

Fatty acid profile of buccal cheek cell phospholipids as an index for dietary intake of docosahexaenoic acid in preterm infants

Cheek cells (buccal epithelia) were utilized as a noninvasive index of fatty acid status in a study of the effects of n−3 long chain polyunsaturated fatty acid supplementation on visual function in preterm infants. The fatty acid profile of cheek cell phospholipids was directly correlated with the dietary docosahexenoic acid (DHA) intake of infants receiving: (i) primarily human milk; (ii) n−3 fatty acid-deficient, corn oil-based, commercial formula (CO); (iii) α-linolenic acid-enriched, soy oilbased, commercial formula; or (iv) experimental formula enriched with soy and marine oils providing a DHA level equivalent to that in human milk. In a subset of infants with complete cheek cell fatty acid profiles and visual function assessments, preterm infants at both 36 wk (n=63) and 57 wk (n=45) postconceptional age had significantly (P<0.0005) reduced cheek cell phospholipid DHA levels in the n−3-dificient, CO-fed group compared to the other diet groups. The DHA content in cheek cell phospholipids was highly correlated (P<0.0005) with that of both red blood cell lipids and plasma phospholipids at the 36-and 57-wk time points. The DHA content in cheek cell lipids of infants at 36 wk was significantly correlated with electroretinographic responses (r=−0.29; P<0.03) and visual acuity (r=−0.31; P<0.02) as measured by visual-evoked potentials (VEP). Cheek cell DHA was highly correlated (r=−0.57; P<0.0005) with VEP acuity at the 57-wk time point. These results suggest that the fatty acid profile of cheek cells is a valid index of essential fatty acid status, can be monitored frequently, and is associated with functional parameters in infants.     

Interaction of CETP inhibitory peptide and lipoprotein substrates in cholesteryl ester transfer assay: relationship between association properties and inhibitory activities

In a previous study, CETP inhibitory peptide (3 kDa) was isolated from hog plasma. The peptide, synthesized chemically according to the amino acid sequence of the 3-kDa peptide (designated P₂₈), showed CETP inhibitory activity both in vitro and in vivo [Cho et al. (1998) Biochim. Biophys. Acta 1391, 133–144]. We report herein further unique features of P₂₈ when it was associated with the cholesteryl ester (CE)-donor and-acceptor lipoproteins. Lipoprotein substrates with P₂₈ present in both HDL (as a CE-donor) and LDL (as a CE-acceptor) served as poor substrates, with CE-transfer activity decreased up to 60% compared to normal substrates without P₂₈. P₂₈ was found to be located in HDL fractions of hog plasma and showed the same electromobility as that visualized by PAGE on 7% polyacrylamide gel under nondenaturing conditions. Addition of apolipoprotein A-1 (apoA-1) or apoB antibody to a normal CE-transfer mixture did not alter CE-transfer activity. However, addition of apoA-1 or −B antibody to a CETP-inhibition mixture decreased the inhibitory activity of P₂₈ by ca. 20%. Western blot analysis revealed that P₂₈ was associated only with human and hog HDL among several lipoproteins purified from human, hog, and rabbit. CFTP-inhibition assays with various lipoprotein substrates revealed that P₂₈ exhibited substrate-specific inhibitory activity. The inhibitory activity of P₂₈ was highly dependent on the type of lipoprotein substrate (whether CE-donor or-acceptor); P₂₈ inhibited CE transfer from HDL to LDL, but it did not inhibit CE transfer from HDL to HDL.     

Trans isomeric octadecenoic acids are related inversely to arachidonic acid and DHA and positively related to mead acid in umbilical vessel wall lipids

Long-chain PUFA play an important role in early human neurodevelopment. Significant inverse correlations were reported between values of trans isomeric and long-chain PUFA in plasma lipids of preterm infants and children aged 1–15 yr as well as in venous cord blood lipids of full-term infants. Here we report FA compositional data of cord blood vessel wall lipids in 308 healthy, full-term infants (gestational age: 39.7±1.2 wk, birth weight: 3528±429 g, mean±SD). The median (interquartile range) of the sum of 18-carbon trans FA was 0.22 (0.13)% w/w in umbilical artery and 0.16 (0.10)% w/w in umbilical vein lipids. Nonparametric correlation analysis showed significant inverse correlations between the sum of 18-carbon trans FA and both arachidonic acid and DHA in artery (r=−0.38, P<0.01, and r=−0.20, P<0.01) and vein (r=−0.36, P<0.01, and −0.17, P<0.01) wall lipids. In addition, the sum of 18-carbon trans FA was significantly positively correlated to Mead acid, a general indicator of EFA deficiency, in both artery (r=+0.35, P<0.01) and vein (r=+0.31, P<0.01) wall lipids. The present results obtained in a large group of full-term infants suggest that maternal trans FA intake is inversely associated with long-chain PUFA status of the infant at birth.     

A significant inverse relationship between concentrations of plasma homocysteine and phospholipid docosahexaenoic acid in healthy male subjects

The aim of this study was to investigate the possibility of a relationship between plasma homocysteine (Hcy) and phospholipid FA (PUFA) in healthy Australian males. One hundred thirty six healthy male subjects aged 20–55 yr were recruited from the Melbourne metropolitan area. Each volunteer completed a semiquantitative food frequency questionnaire and gave a blood sample. Plasma Hcy concentrations were determined by an established HPLC method; the plasma phospholipid FA were determined by standard methods. Plasma Hcy concentration was significantly negatively correlated with plasma phospholipid concentration of the PUFA 20∶5n−3 (r=−0.226, P=0.009), 22∶5n−3 (r=−0.182, P=0.036), 22∶6n−3 (r=−0.286, P=0.001), total n−3 (r=−0.270, P=0.002) and the ratio n−3/n−6 PUFA (r=−0.265, P=0.002), and significantly positively correlated with 20∶4n−6 (r=0.180, P=0.037). In the partial correlation analysis, after controlling for serum vitamin B₁₂ and folate concentration, plasma Hcy was significantly negatively correlated with the plasma phospholipid concentration of 22∶6n−3 (r=−0.205, P=0.019), total n−3 (r=−0.182, P=0.038) and the ratio n−3/n−6 PUFA (r=−0.174, P=0.048). Evidence indicates that an increased concentration of n−3 PUFA in tissues has a beneficial effect on cardiovascular health. Our findings provide further evidence that increased consumption of dietary n−3 PUFA increases the concentration of n−3 PUFA in plasma phospholipid, which is associated with a protective effect on cardiovascular diseases and lower plasma Hcy levels. The mechanism that might explain the association between plasma 22∶6n−3 and Hcy levels is not clear.     

Postprandial and short-term effects of dietary virgin olive oil on oxidant/antioxidant status

It is generally believed that virgin olive oil consumption has beneficial effects, but little is known about its effects postprandially on oxidant/antioxidant status. The aim of this study was to determine changes in oxidative stress biomarkers and lipid profile after a single dose of virgin olive oil and after 1 wk of daily consumption. Sixteen subjects (9 men, 7 women) ingested 50 mL of virgin olive oil in a single dose. Blood samples were collected from 0 to 24 h. Thereafter, 14 participants (8 men, 6 women) followed a 1-wk 25 mg/d virgin olive oil dietary intervention. Blood samples were collected at the end of this period. Serum TAG (P=0.016), plasma FA (P<0.001) and lipid peroxidation products in plasma (P<0.001) and VLDL (P=0.007) increased, reaching a peak at 4–6 h, and returning to baseline values at 24 h after oil ingestion. The opposite changes were observed in plasma glutathione peroxidase (P=0.001) and glutathione reductase (GR) (P=0.042). No changes in LDL lipid peroxidation or resistance to oxidation were observed postprandially. At 24 h, plasma oleic acid remained increased (P<0.05) and resistance of LDL to oxidation improved (P<0.05). After 1 wk of virgin olive oil consumption, plasma oleic acid (P=0.031), resistance of LDL to oxidation (P<0.05), and plasma GR activity (P=0.005) increased. These results indicate that changes in oxidant/antioxidant status occur after oral virgin olive oil. Virgin olive oil consumption could provide short-term benefits for LDL resistance to oxidation and in glutathione-related enzyme activities.