Protein and lipid binding parameters in rainbow trout (Oncorhynchus mykiss) blood and liver fractions to extrapolate from an in vitro metabolic degradation assay to in vivo bioaccumulation potential of hydrophobic organic chemicals

Binding of hydrophobic chemicals to colloids such as proteins or lipids is difficult to measure using classical microdialysis methods due to low aqueous concentrations, adsorption to dialysis membranes and test vessels, and slow kinetics of equilibration. Here, we employed a three-phase partitioning system where silicone (polydimethylsiloxane, PDMS) serves as a third phase to determine partitioning between water and colloids and acts at the same time as a dosing device for hydrophobic chemicals. The applicability of this method was demonstrated with bovine serum albumin (BSA). Measured binding constants (K(BSAw)) for chlorpyrifos, methoxychlor, nonylphenol, and pyrene were in good agreement with an established quantitative structure-activity relationship (QSAR). A fifth compound, fluoxypyr-methyl-heptyl ester, was excluded from the analysis because of apparent abiotic degradation. The PDMS depletion method was then used to determine partition coefficients for test chemicals in rainbow trout (Oncorhynchus mykiss) liver S9 fractions (K(S9w)) and blood plasma (K(bloodw)). Measured K(S9w) and K(bloodw) values were consistent with predictions obtained using a mass-balance model that employs the octanol-water partition coefficient (K(ow)) as a surrogate for lipid partitioning and K(BSAw) to represent protein binding. For each compound, K(bloodw) was substantially greater than K(S9w), primarily because blood contains more lipid than liver S9 fractions (1.84% of wet weight vs 0.051%). Measured liver S9 and blood plasma binding parameters were subsequently implemented in an in vitro to in vivo extrapolation model to link the in vitro liver S9 metabolic degradation assay to in vivo metabolism in fish. Apparent volumes of distribution (V(d)) calculated from the experimental data were similar to literature estimates. However, the calculated binding ratios (f(u)) used to relate in vitro metabolic clearance to clearance by the intact liver were 10 to 100 times lower than values used in previous modeling efforts. Bioconcentration factors (BCF) predicted using the experimental binding data were substantially higher than the predicted values obtained in earlier studies and correlated poorly with measured BCF values in fish. One possible explanation for this finding is that chemicals bound to proteins can desorb rapidly and thus contribute to metabolic turnover of the chemicals. This hypothesis remains to be investigated in future studies, ideally with chemicals of higher hydrophobicity.

Intramyocellular lipid variations in active older men: relationship with aerobic fitness

Intramyocellular lipid (IMCL) variations in older men are poorly explored. In young adults, IMCL can be influenced by both diet and exercise interventions; this flexibility is related to aerobic fitness. We evaluated in active older adults the influence of maximal aerobic capacity on short-term diet and exercise-induced variations in IMCL stores.

The lipid transporters ABCA1 and ABCG1 are differentially expressed in syncytiotrophoblasts of preeclamptic and IUGR placentas

A91

Perilipin-1 in hemodialyzed patients: association with history of coronary heart disease and lipid profile

Perilipin-1 surrounds lipid droplets in both adipocytes and in atheroma plaque foam cells and controls access of lipases to the lipid core. In hemodialysis (HD) patients, dyslipidemia, malnutrition, inflammation and atherosclerosis are common. Thirty-six HD patients and 28 healthy volunteers were enrolled into the study. Ten HD patients suffered from coronary heart disease (CHD). Perilipin-1, triglycerides, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol (HDL-C), body mass index, albumin, geriatric nutritional risk index, normalized protein catabolic rate, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured. Perilipin-1 did not differ between HD patients and healthy volunteers. IL-6 and TNF-α were higher in HD patients. The evaluated nutritional markers and the markers of inflammation did not differ between HD patients with high perilipin-1 levels and HD patients with low perilipin-1 levels. Regarding the lipid profile, only HDL-C differed between HD patients with high perilipin-1 levels and HD patients with low perilipin-1 levels, and it was higher in the first subgroup. Perilipin-1 was significantly higher in HD patients without CHD. Perilipin-1 is detectable in the serum of HD patients and it is associated with increased HDL-C and decreased incidence of CHD.

Association of major and minor ECG abnormalities with coronary heart disease events

In populations of older adults, prediction of coronary heart disease (CHD) events through traditional risk factors is less accurate than in middle-aged adults. Electrocardiographic (ECG) abnormalities are common in older adults and might be of value for CHD prediction.

Different duration of at-risk mental state associated with neurofunctional abnormalities. A multimodal imaging study

Objectives: Neurofunctional alterations are correlates of vulnerability to psychosis, as well as of the disorder itself. How these abnormalities relate to different probabilities for later transition to psychosis is unclear. We investigated vulnerability- versus disease-related versus resilience biomarkers of psychosis during working memory (WM) processing in individuals with an at-risk mental state (ARMS). Experimental design: Patients with “first-episode psychosis” (FEP, n = 21), short-term ARMS (ARMS-ST, n = 17), long-term ARMS (ARMS-LT, n = 16), and healthy controls (HC, n = 20) were investigated with an n-back WM task. We examined functional magnetic resonance imaging (fMRI) and structural magnetic resonance imaging (sMRI) data in conjunction using biological parametric mapping (BPM) toolbox. Principal observations: There were no differences in accuracy, but the FEP and the ARMS-ST group had longer reaction times compared with the HC and the ARMS-LT group. With the 2-back > 0-back contrast, we found reduced functional activation in ARMS-ST and FEP compared with the HC group in parietal and middle frontal regions. Relative to ARMS-LT individuals, FEP patients showed decreased activation in the bilateral inferior frontal gyrus and insula, and in the left prefrontal cortex. Compared with the ARMS-LT, the ARMS-ST subjects showed reduced activation in the right inferior frontal gyrus and insula. Reduced insular and prefrontal activation was associated with gray matter volume reduction in the same area in the ARMS-LT group. Conclusions: These findings suggest that vulnerability to psychosis was associated with neurofunctional alterations in fronto-temporo-parietal networks in a WM task. Neurofunctional differences within the ARMS were related to different duration of the prodromal state and resilience factors

Insular volume abnormalities associated with different transition probabilities to psychosis

Background Although individuals vulnerable to psychosis show brain volumetric abnormalities, structural alterations underlying different probabilities for later transition are unknown. The present study addresses this issue by means of voxel-based morphometry (VBM). Method We investigated grey matter volume (GMV) abnormalities by comparing four neuroleptic-free groups: individuals with first episode of psychosis (FEP) and with at-risk mental state (ARMS), with either long-term (ARMS-LT) or short-term ARMS (ARMS-ST), compared to the healthy control (HC) group. Using three-dimensional (3D) magnetic resonance imaging (MRI), we examined 16 FEP, 31 ARMS, clinically followed up for on average 3 months (ARMS-ST, n=18) and 4.5 years (ARMS-LT, n=13), and 19 HC. Results The ARMS-ST group showed less GMV in the right and left insula compared to the ARMS-LT (Cohen's d 1.67) and FEP groups (Cohen's d 1.81) respectively. These GMV differences were correlated positively with global functioning in the whole ARMS group. Insular alterations were associated with negative symptomatology in the whole ARMS group, and also with hallucinations in the ARMS-ST and ARMS-LT subgroups. We found a significant effect of previous antipsychotic medication use on GMV abnormalities in the FEP group. Conclusions GMV abnormalities in subjects at high clinical risk for psychosis are associated with negative and positive psychotic symptoms, and global functioning. Alterations in the right insula are associated with a higher risk for transition to psychosis, and thus may be related to different transition probabilities.

PP141. The lipid transporters ABCA1 and ABCG1 are differentially expressed in preeclamptic and IUGR placentas

INTRODUCTION The ATP-binding cassette (ABC) transporter A1 (ABCA1) and ABCG1 are highly expressed in the placenta in various compartments, including the villous syncytiotrophoblast (V-STB) and foetal endothelial cells. Among other not yet characterized functions, they play a role in the foeto-maternal transport of cholesterol and other lipophilic molecules. In humans, preliminary data suggest expressional changes of ABCA1 and ABCG1 in pathologic gestation, particularly under hypoxic conditions, but a systematic expression analysis in common human pregnancy diseases has never been performed. OBJECTIVES The aim of the present study was to characterize ABCA1 and ABCG1 expression in a large series of pathologic placentas, in particular from preeclampsia (PE) and intrauterine growth restriction (IUGR) which are associated with placental hypoxia. METHODS Placentas from 152 pathological pregnancies, including PE and/or HELLP (n=24) and IUGR (n=21), and 20 normal control placentas were assessed for their ABCA1 and ABCG1 mRNA and protein expression with quantitative RT-PCR and semi-quantitative immunohistochemical analysis, respectively. RESULTS ABCA1 protein expression in the V-STB was significantly less extensive in PE compared with normal controls (<10% of V-STB stained for ABCA1 in 58% PE placentas vs. 25% controls; p=0.035). Conversely, it was significantly more wide-spread in IUGR (>75% of V-STB stained in 57% IUGR placentas vs. 15% controls; p=0.009). Moreover, there was an insignificant trend for increased ABCA1 expression in fetal endothelial cells of stem villi in PE (p=0.0588). ABCA1 staining levels in V-STB were significantly associated with placental histopathological features related with hypoxia: they were decreased in placentas exhibiting syncytial knotting (p=0.033) and decidual vasculopathy (p=0.0437) and increased in low weight placentas (p=0.015). The significant and specific alterations in ABCA1 protein expression found at a specific cellular level were not paralleled by changes in ABCA1 mRNA abundance of total placental tissue. ABCG1 staining was universally extensive in the V-STB of normal placentas, always affecting more than 90% of V-STB surface. In comparison, ABCG1 staining of the V-STB was generally often reduced in pregnancy diseases. In particular, less than 90% of V-STB exhibited ABCG1 staining in 26% of PE placentas (p=0.022) and 35% of IUGR placentas (p=0.003). Similarly to ABCA1, ABCG1 mRNA expression in total placental tissue was not significantly different between controls and PE or IUGR. CONCLUSION ABCA1 and ABCG1 proteins are differentially expressed, with either down- or up-regulation, in the V-STB of placentas exhibiting features of chronic hypoxia, such as in PE and IUGR. This suggests that other factors in addition to hypoxia regulate the expression of placental lipid transporters. The specific changes on a cellular level were masked when only total tissue mRNA was analysed underlining the importance of cell specific expression analysis. The potential effects of decreased placental ABCA1 and ABCG1 expression on foetal nutrition and development remain to be elucidated.

[Determination of maternal and foetal serum lipid profile and placental oxidised low density lipoprotein accumulation in preeclampsia and normotensive pregnancies]

Oxidised low density lipoproteins (oxLDL) are key players in the development of atherosclerotic cardiovascular diseases. Since there are similarities between the pathogenesis of preeclampsia and atherosclerosis we hypothesised an increased accumulation of oxLDL at the materno-foetal and foeto-foetal interface within the placental tissue of preeclamptic women compared to women with normotensive pregnancies (controls). Moreover, we analysed maternal and foetal serum lipid parameters.

Hypoxia and lipid signaling

Sufficient oxygen supply is crucial for the development and physiology of mammalian cells and tissues. When simple diffusion of oxygen becomes inadequate to provide the necessary flow of substrate, evolution has provided cells with tools to detect and respond to hypoxia by upregulating the expression of specific genes, which allows an adaptation to hypoxia-induced stress conditions. The modulation of cell signaling by hypoxia is an emerging area of research that provides insight into the orchestration of cell adaptation to a changing environment. Cell signaling and adaptation processes are often accompanied by rapid and/or chronic remodeling of membrane lipids by activated lipases. This review highlights the bi-directional relation between hypoxia and lipid signaling mechanisms.

Posttranscriptional regulation of Fas (CD95) ligand killing activity by lipid rafts

Fas (CD95/Apo-1) ligand-mediated apoptosis induction of target cells is one of the major effector mechanisms by which cytotoxic lymphocytes (T cells and natural killer cells) kill their target cells. In T cells, Fas ligand expression is tightly regulated at a transcriptional level through the activation of a distinct set of transcription factors. Increasing evidence, however, supports an important role for posttranscriptional regulation of Fas ligand expression and activity. Lipid rafts are cholesterol- and sphingolipid-rich membrane microdomains, critically involved in the regulation of membrane receptor signaling complexes through the clustering and concentration of signaling molecules. Here, we now provide evidence that Fas ligand is constitutively localized in lipid rafts of FasL transfectants and primary T cells. Importantly, disruption of lipid rafts strongly reduces the apoptosis-inducing activity of Fas ligand. Localization to lipid rafts appears to be predominantly mediated by the characteristic cytoplasmic proline-rich domain of Fas ligand because mutations of this domain result in reduced recruitment to lipid rafts and attenuated Fas ligand killing activity. We conclude that Fas ligand clustering in lipid rafts represents an important control mechanism in the regulation of T cell-mediated cytotoxicity.

Statin therapy induces ultrastructural damage in skeletal muscle in patients without myalgia

Muscle pain and weakness are frequent complaints in patients receiving 3-hydroxymethylglutaryl coenzymeA (HMG CoA) reductase inhibitors (statins). Many patients with myalgia have creatine kinase levels that are either normal or only marginally elevated, and no obvious structural defects have been reported in patients with myalgia only. To investigate further the mechanism that mediates statin-induced skeletal muscle damage, skeletal muscle biopsies from statin-treated and non-statin-treated patients were examined using both electron microscopy and biochemical approaches. The present paper reports clear evidence of skeletal muscle damage in statin-treated patients, despite their being asymptomatic. Though the degree of overall damage is slight, it has a characteristic pattern that includes breakdown of the T-tubular system and subsarcolemmal rupture. These characteristic structural abnormalities observed in the statin-treated patients were reproduced by extraction of cholesterol from skeletal muscle fibres in vitro. These findings support the hypothesis that statin-induced cholesterol lowering per se contributes to myocyte damage and suggest further that it is the specific lipid/protein organization of the skeletal muscle cell itself that renders it particularly vulnerable.

Intramyocellular lipid stores increase markedly in athletes after 1.5 days lipid supplementation and are utilized during exercise in proportion to their content

Intramyocellular lipids (IMCL) and muscle glycogen provide local energy during exercise (EX). The objective of this study was to clarify the role of high versus low IMCL levels at equal initial muscle glycogen on fuel selection during EX. After 3 h of depleting exercise, 11 endurance-trained males consumed in a crossover design a high-carbohydrate (7 g kg(-1) day(-1)) low-fat (0.5 g kg(-1) day(-1)) diet (HC) for 2.5 days or the same diet with 3 g kg(-1) day(-1) more fat provided during the last 1.5 days of diet (four meals; HCF). Respiratory exchange, thigh muscle substrate breakdown by magnetic resonance spectroscopy, and plasma FFA oxidation ([1-(13)C]palmitate) were measured during EX (3 h, 50% W (max)). Pre-EX IMCL concentrations were 55% higher after HCF. IMCL utilization during EX in HCF was threefold greater compared with HC (P < 0.001) and was correlated with aerobic power and highly correlated (P < 0.001) with initial content. Glycogen values and decrements during EX were similar. Whole-body fat oxidation (Fat(ox)) was similar overall and plasma FFA oxidation smaller (P < 0.05) during the first EX hour after HCF. Myocellular fuels contributed 8% more to whole-body energy demands after HCF (P < 0.05) due to IMCL breakdown (27% Fat(ox)). After EX, when both IMCL and glycogen concentrations were again similar across trials, a simulated 20-km time-trial showed no difference in performance between diets. In conclusion, IMCL concentrations can be increased during a glycogen loading diet by consuming additional fat for the last 1.5 days. During subsequent exercise, IMCL decrease in proportion to their initial content, partly in exchange for peripheral fatty acids.

The effect of increased lipid intake on hormonal responses during aerobic exercise in endurance-trained men

In view of the growing health problem associated with obesity, clarification of the regulation of energy homeostasis is important. Peripheral signals, such as ghrelin and leptin, have been shown to influence energy homeostasis. Nutrients and physical exercise, in turn, influence hormone levels. Data on the hormonal response to physical exercise (standardized negative energy balance) after high-fat (HF) or low-fat (LF) diet with identical carbohydrate intake are currently not available. The aim of the study was to investigate whether a short-term dietary intervention with HF and LF affects ghrelin and leptin levels and their modulators, GH, insulin and cortisol, before and during aerobic exercise. Eleven healthy, endurance-trained male athletes (W(max) 365 +/- 29 W) were investigated twice in a randomized crossover design following two types of diet: 1. LF - 0.5 g fat/kg body weight (BW) per day for 2.5 days; 2. HF - 0.5 g fat/kg BW per day for 1 day followed by 3.5 g fat/kg BW per day for 1.5 days. After a standardized carbohydrate snack in the morning, metabolites and hormones (GH, ghrelin, leptin, insulin and cortisol) were measured before and at regular intervals throughout a 3-h aerobic exercise test on a cycloergometer at 50% of W(max). Diet did not significantly affect GH and cortisol concentrations during exercise but resulted in a significant increase in ghrelin and decrease in leptin concentrations after LF compared with HF diet (area under the curve (AUC) ghrelin LF vs HF: P < 0.03; AUC leptin LF vs HF: P < 0.02, Wilcoxon rank test). These data suggest that acute negative energy balance induced by exercise elicits a hormonal response with opposite changes of ghrelin and leptin. In addition, the hormonal response is modulated by the preceding intake of fat.

Role of proton MR for the study of muscle lipid metabolism

1H-MR spectroscopy (MRS) of intramyocellular lipids (IMCL) became particularly important when it was recognized that IMCL levels are related to insulin sensitivity. While this relation is rather complex and depends on the training status of the subjects, various other influences such as exercise and diet also influence IMCL concentrations. This may open insight into many metabolic interactions; however, it also requires careful planning of studies in order to control all these confounding influences. This review summarizes various historical, methodological, and practical aspects of 1H-MR spectroscopy (MRS) of muscular lipids. That includes a differentiation of bulk magnetic susceptibility effects and residual dipolar coupling that can both be observed in MRS of skeletal muscle, yet affecting different metabolites in a specific way. Fitting of the intra- (IMCL) and extramyocellular (EMCL) signals with complex line shapes and the transformation into absolute concentrations is discussed. Since the determination of IMCL in muscle groups with oblique fiber orientation or in obese subjects is still difficult, potential improvement with high-resolution spectroscopic imaging or at higher field strength is considered. Fat selective imaging is presented as a possible alternative to MRS and the potential of multinuclear MRS is discussed. 1H-MRS of muscle lipids allows non-invasive and repeated studies of muscle metabolism that lead to highly relevant findings in clinics and patho-physiology.