Metabolomics (liver and blood profiling) in a mouse model in response to fasting: a study of hepatic steatosis.

A metabolomic approach was applied to a mouse model of starvation-induced hepatic steatosis. After 24 h of fasting it appears that starvation reduced the phospholipids (PL), free cholesterol (FC), and cholesterol esters (CE) content of low-density lipoproteins (LDL). In liver lipid profiles major changes were observed using different techniques. High performance thin layer chromatography (HPTLC)-measurements of liver-homogenates indicated a significant rise of FC with 192%, triacylglycerols (TG) with 456% and cholesterol esters (CE) with 268% after 24 h of starvation in comparison with the control group. Reversed phase liquid chromatography coupled to mass spectrometry measurements (LC-MS) of liver homogenate indicated that the intensity of Phosphatidylcholine (PC) in the 24-h starvation group dropped to 90% of the value in the control group while the intensity of CE and TG increased to 157% and 331%, respectively, of the control group. Interestingly, a 49:4-TG with an odd number of C atoms appeared during starvation. This unique triacylglycerol has all characteristics of a biomarker for detection of hepatic steatosis. These observations indicate that in mammals liver lipid profiles are a dynamic system which are readily modulated by environmental factors like starvation.

Does a 5500-km swim trial stimulate early sexual maturation in the European eel (Anguilla anguilla L.)?

The catadromous European eel (Anguilla anguilla L.) undertakes a 6000-km spawning migration from its freshwater habitats to the Sargasso Sea. In large Blazka swim tunnels of 127 l, the physiological effect of such a prolonged swimming performance on sexual maturation in adult female eels was investigated. Two groups of eels were placed in swim tunnels for 173 days, one group was able to swim at 0.5 body lengths/second (Swim group) covering a distance of c. 5500-km over the experimental period, and one group kept in static (End Control group). A control group was sampled at the start of the experiment in order to determine the initial stage of reproductive development (Initial Control group). At the end of the swim trial, the maturation parameters 11-ketotestosterone, pituitary levels of LH and plasma levels of estradiol were higher (although not significantly) in the Swim compared to the End Control group. In addition, no significant differences were observed in most measured morphometric and reproductive parameters, including eye-index, gonadosomatic index, hepatosomatic index, and plasma levels of vitellogenin, cortisol and melanophore-stimulating hormone (MSH). Also, pituitary levels of both MSH, and adrenocorticotropic hormone (ACTH) were unaffected. In contrast, the oocyte diameter was found to be significantly higher in the Swim compared to the End Control group. Based on these observations we conclude that a period of prolonged swimming might be a physiological stimulus necessary for the onset of maturation in the European eel.

Surgical procedure for implanting a radiotelemetry transmitter to monitor ECG, heart rate and body temperature in small Carassius auratus and Carassius auratus gibelio under laboratory conditions.

Radiotelemetry provides an alternative means of obtaining physiological measurements from conscious and freely moving animals, without introducing stress artefacts. A surgical procedure is described for implanting radiotelemetry transmitters to monitor the electrocardiogram (ECG), heart rate (HR) and body temperature (BT) in small goldfish (Carassius auratus; 50-100 g) and Prussian carp (Carassius auratus gibelio; 100 g). This type of transmitter is commonly implanted in freely moving mice. After surgery and a recovery period of 24 h, the ECG, HR and BT were recorded in freely swimming fish within the limitations of the aquarium.

Are dioxin-like contaminants responsible for the eel (Anguilla anguilla) drama?

Eel populations worldwide are dangerously close to collapsing. Our study is the first to show that current levels of dioxin-like contaminants are strong candidates because of their devastating effects on development and survival of eel embryos. Female and male silver eels were artificially stimulated to maturation and reproduction by treatment with carp pituitary extracts and hCG, respectively. During maturation of female European silver eels, about 60 g fat per kg eel is incorporated in the oocytes. Together with the fat, however, persistent organic pollutants such as dioxin-like polychlorinated biphenyls (PCBs) are incorporated too. The total dioxin-like toxic potency of the individual gonad batches was determined as 2,3,7,8-tetrachlorodibenzo-p-dioxine equivalents (TEQs), using an in vitro reporter gene assay. The observed differences in development and survival showed a significant negative correlation with the TEQ levels in the gonads, already at levels far below the maximal allowable level for fish consumption, i.e., 4 ng TEQ/kg fish. The clear inverse relationship between the TEQ level and the survival period of the fertilised eggs strongly suggests that the current levels of dioxin-like compounds seriously impair the reproduction of the European eel. The peak of the environmental levels of dioxin-like PCBs and the decline of eel coincide worldwide, further suggesting that, in addition to other threats, these contaminants contributed significantly to the current collapse in eel populations.

Hematology patterns of migrating European eels and the role of EVEX virus.

We show that European eels infected with the rhabdovirus EVEX (Eel Virus European X) virus, developed hemorrhage and anemia during simulated migration in large swim tunnels, and died after 1000-1500 km. In contrast, virus-negative animals swam 5500 km, the estimated distance to the spawning ground of the European eel in the Sargasso Sea. Virus-positive eels showed a decline in hematocrit, which was related to the swim distance. Virus-negative eels showed a slightly increased hematocrit. Observed changes in plasma lactate dehydrogenase (LDH), total protein and aspartate aminotransferase (AAT) are indicative of a serious viral infection. Based on these observations, we conclude that eel virus infections may adversely affect the spawning migration of eels, and could be a contributing factor to the worldwide decline of eel.

Phosphorylation state of red and white muscle in tilapia during graded hypoxia: an in vivo (31)P-NMR study.

The aim of this study was to measure the energetic consequences of hypoxia in different types of skeletal muscle within a single tilapia species (n = 5). To that aim, 81.0 MHz (31)P-nuclear magnetic resonance (NMR) spectra were collected, alternately, from three surface coils placed adjacent to the tissues of interest (dorsal white muscle, ventral white muscle, and lateral red muscle) during a graded hypoxia load over 6 h followed by a 5-h recovery period. The fish were contained in a flow cell, enabling us full control of the oxygen content of the bathing medium. The intracellular pH and the concentrations of ATP, phosphocreatine (PCr), and P(i) were determined from the NMR spectra. For normoxia, biochemical differences for [gamma-ATP], [PCr], and [sugar phosphates] (SP) were observed between all three locations, especially between the red and white muscle. During hypoxia stress, loss of phosphorylated compounds (PCr+P(i)+SP) was observed at all locations but was the most severe in red muscle. When the aerobic (respirometry) and anaerobic ((31)P-NMR) ATP production via an energy balance are compared, flexible metabolic depression is demonstrated during anaerobioses. It is concluded that control of the aerobic and anaerobic component of metabolism during metabolic depression is independent of each other.

Synergistic effect of acidification and hypoxia: in vivo 31P-NMR and respirometric study in fishes.

We used 31P nuclear magnetic resonance (31P-NMR) spectroscopy to measure intracellular pH (pHi) and high-energy phosphate levels of white muscle of the fish tilapia (Oreochromis mossambicus) during exposure to the stressors hypoxia and water acidification separately and simultaneously. The protocol for the graded hypoxia load was 100, 40, 30, 20, 10, 5, and 3% air saturation for 1 h at each level. For environmental acidosis the pH of the water was lowered from 7.6 to 4.0 over 6 h, with time of exposure approximately 11 h. All protocols were followed by 6 h of reoxygenation at 20 degrees C. We also measured total oxygen consumption of the animal. The results of this in vivo study revealed that environmental acidification had no effect on oxygen consumption, pHi, or phosphocreatine depletion. Hypoxia caused moderate changes in these parameters and fast and complete recovery during reoxygenation. In contrast, the combination of environmental acidosis and hypoxia resulted in 50% mortality, increased depletion of the phosphocreatine pool, and a retarded recovery of the pHi during reoxygenation compared with the group with hypoxia as a single stressor. The combination of environmental acidification and hypoxia has a more profound effect and works synergistically compared with the conditions imposed separately. To investigate whether an adaptation response occurred during chronic exposure to environmental acidification, animals were exposed for 6 wk to pH 4.0 before experimentation. The pHi in the white muscle dropped from 7.2 (control group) to 6.9 during this period, whereas no effect was found in the phosphorylated compounds and oxygen consumption. Therefore, it is concluded that no adaptation response occurs in animals exposed to long-term environmental acidosis.

Fish muscle energy metabolism measured during hypoxia and recovery: an in vivo 31P-NMR study.

Three fish species were exposed to graded hypoxia levels and allowed to recover. Levels of high-energy phosphate compounds in epaxial white muscle were monitored by in vivo 31P nuclear magnetic resonance (NMR) spectroscopy. Furthermore, O2 consumption of the animals was measured. With increasing hypoxia load, metabolic parameters started to change in the following order: phosphocreatine (PCr)-to-Pi ratio (decrease), O2 consumption (decrease), [PCr] (decrease), intracellular pH (pHi; decrease), Pi (increase), free ADP concentration ([ADP]free; increase), [ATP] (decrease). PCr levels fell with the PO2. After each increment, the [PCr] reached a stable plateau value while, in some cases, a recovery was observed. This recovery could be explained because the balance between anaerobic and aerobic metabolism is continuously fluctuating during hypoxia as a consequence of changes in the activity of the fish. Consequently the [ADP]free are fluctuating, resulting in an activation of the creatine kinase reaction and the anaerobic glycolysis. In all three species, anaerobic glycolysis was activated, but in contrast to anoxia exposure, metabolic suppression was absent. The changes of [ADP]free and [H+] (which influences the position of the creatine kinase equilibrium) are species dependent. Species differences observed in the other parameters were small. It is concluded that the pattern of the activation of anaerobic metabolism under deep hypoxia is different from that under anoxia.

Pharmacokinetics of sulphadimidine in carp (Cyprinus carpio L.) and rainbow trout (Salmo gairdneri Richardson) acclimated at two different temperature levels.

The influence of temperature (10 degrees C and 20 degrees C) on pharmacokinetics and metabolism of sulphadimidine (SDM) in carp and trout was studied. At 20 degrees C a significantly lower level of distribution (Vdarea) and a significantly shorter elimination half-life (T(1/2)beta) was achieved in both species compared to the 10 degrees C level. In carp the body clearance parameter (ClB(SDM)) was significantly higher at 20 degrees C compared to the value at 10 degrees C, whereas for trout this parameter was in the same order of magnitude for both temperatures. N4-acetylsulphadimidine (N4-SDM) was the main metabolite of SDM in both species at the two temperature levels. The relative N4-SDM plasma percentage in carp was significantly higher at 20 degrees C than at 10 degrees C, whereas there was in trout no significant difference. In neither species was the peak plasma concentration of N4-SDM (Cmax(N4-SDM)) significantly different at two temperatures. The corresponding peak time of this metabolite (Tmax(N4-SDM)) was significantly shorter at 20 degrees C compared to 10 degrees C in both carp and trout. In carp at both temperatures, acetylation occurs to a greater extent than hydroxylation. Only the 6-hydroxymethyl-metabolite (SCH2OH) was detected in carp, at a significant different level at the two temperatures. Concentrations of hydroxy metabolites in trout were at the detection level of the HPLC-method (0.02-micrograms/ml). The glucuronide metabolite (SOH-gluc.) was not detected in either species at the two temperatures.