Molecular biomarkers in grey seals (Halichoerus grypus) to evaluate pollutant exposure, health and immune status.

Grey seals as top-predators bioaccumulate contaminants and can be considered as sentinels of eco-system health. Pups are weaned after a short nursing period, characterised by an enormous lipid transfer and exposure to contaminants. This study established molecular biomarkers of the xenobiotic metabolism and immune system to help assess health and immune status. mRNA transcription of AHR, ARNT, PPARα and cytokine IL-2 and heat-shock-protein HSP70 was measured in blood of grey seal pups and adults in rehabilitation and permanent care using RT-qPCR and compared to rehabilitating harbour seal pups and haematology values. In pups highest levels at admission in xenobiotic biomarker, HSP70 and cytokine transcription may show contaminant exposure via lactation, stress during abandonment and dehydration. The significant decrease may be linked to diet, health improvement and adaptation. Adults showed higher levels and more variation in biomarker transcription and clear species-specific differences between harbour and grey seal pups were found.

Variable transcription of pro- and anti-inflammatory cytokines in phocine lymphocytes following canine distemper virus infection.

Canine distemper virus (CDV) is a highly contagious viral pathogen. Domesticated dogs are the main reservoir of CDV. Although phocine distemper virus was responsible for the recent epidemics in seals in the North and Baltic Seas, most devastating epidemics in seals were also caused by CDV. To further study the pathogenesis of CDV infection in seals, it was the aim of the present study to investigate the mechanisms of CDV induced immunosuppression in seals by analyzing the gene transcription of different pro- and anti-inflammatory cytokines in Concanavalin A (Con A) stimulated and non-stimulated phocine lymphocytes in vitro following infection with the CDV Onderstepoort (CDV-OND) strain. Phocine lymphocytes were isolated via density gradient centrifugation. The addition of 1 µg/ml Con A and virus was either performed simultaneously or lymphocytes were stimulated for 48 h with Con A prior to virus infection. Gene transcription of interleukin (IL)-6, IL-12 and tumor necrosis factor alpha (TNFα) as pro-inflammatory cytokines and IL-4, IL-10 and transforming growth factor beta (TGFß) as anti-inflammatory cytokines were determined by using RT-qPCR. CDV-OND infection caused an initial increase of pro-inflammatory phocine cytokines mRNA 24h after infection, followed by a decrease in gene transcription after 48 h. A strong increase in the transcription of IL-4 and TGFß was detected after 48 h when virus and mitogen were added simultaneously. An increased IL-10 production occurred only when stimulation and infection were performed simultaneously. Furthermore, an inhibition of IL-12 on IL-4 was noticed in phocine lymphocytes which were stimulated for 48 h prior to infection. In summary, the duration of the stimulation or the lymphocytes seem to have an important influence on the cytokine transcription and indicates that the outcome of CDV infection is dependent on various factors that might sensitize lymphocytes or make them more susceptible or reactive to CDV infection.

Reversible reproductive control in harbour seals (Phoca vitulina) with a gonadotropin-releasing hormone agonist.

Reproductive control in captive pinnipeds is an important management subject for many facilities. To date reproductive control in harbour seals (Phoca vitulina) has been achieved using anti-androgens, progestagen preparations, castration, and physical separation of the sexes. The harbour seal group at the seal station in Friedrichskoog, Germany consists of three mature females (all >10 years), one older mature male (13 years of age in 2000) and one male who reached maturity during the study (3 years of age in 2000). In 2000 the older mature male received for the first time a 3-month depot injection of a gonadotropin-releasing hormone agonist (buserelin acetetate, 9.9 mg) by subcutaneous injection. This male was subsequently given the gonadotropin-releasing hormone agonist in 2001, 2004 and 2005. The younger male reached maturity during the investigation and received burserelin for the first time in 2004 and again in 2005. No pups were born in 2001, 2002, 2005 or 2006. No reproductive control was performed in 2002 and 2003, resulting in three newborns in 2003 and 2004. Serum levels of testosterone were measured by a routine liquid chromatography coupled with mass spectometry. Pre-burserelin testosterone levels varied between 0.02 and 2.18 ng/ml. Post-burserelin levels were under the detection limit except for the first year of the investigation. No behavioural changes such as changes in social ranking and no clinical side effects were observed. This study shows that the gonadotropin-releasing hormone agonist, burserelin acetate, can be used for reversible reproductive control in harbour seals without observed side effects or detrimental behavioural changes.

Parapoxvirus infection in harbor seals (Phoca vitulina) from the German North Sea.

In the summer of 2000, proliferative lesions of the skin and oral mucosa were observed in 26 young harbor seals (Phoca vitulina) from a rehabilitation center in Schleswig-Holstein, Germany. Verrucose, roundish nodules, approximately 1-2 cm in diameter, were presented in the oral cavity, especially on the tongue. Some animals developed similarly sized spherical dermal elevations with ulceration on flippers, chest, neck, and perineum. Necropsy of one animal showed multifocal, verrucose nodules in the oral cavity and a mild tonsillitis. Histologically, the nodules were characterized by ballooning degeneration of the outer parts of the spiny layer and stratum granulosum, with large eosinophilic cytoplasmic inclusions and a perivascular to interstitial lymphohistiocytic infiltration accompanied by fibroblastic proliferation and neovascularization. Negative staining of mucosal tissue homogenates demonstrated parapoxvirus-like particles. The presence of parapoxvirus was confirmed by polymerase chain reaction, using primers specific for parapoxvirus of ungulates. By in situ hybridization, using a parapox-specific, digoxigenin-labeled DNA probe, abundant parapoxvirus DNA-positive epithelial cells were detected in the stratum granulosum and the outer parts of the spiny layer. There was no parapoxvirus-positive signal in the adjacent submucosa. Although DNA analysis revealed that the causative agent can clearly be distinct from terrestrial parapoxviruses, lesions resembled parapoxvirus infections in other terrestrial species, and the pattern of virus DNA distribution indicated a direct effect of the virus on keratinocytes. In contrast, changes in the corium may be considered an indirect response mediated by the virus or the immune system.

Chronic lithium downregulates cyclooxygenase-2 activity and prostaglandin E(2) concentration in rat brain.

Rats treated with lithium chloride for 6 weeks have been reported to demonstrate reduced turnover of arachidonic acid (AA) in brain phospholipids, and decreases in mRNA and protein levels, and enzyme activity, of AA-selective cytosolic phospholipase A(2)(cPLA(2)). We now report that chronic lithium administration to rats significantly reduced the brain protein level and enzyme activity of cyclooxygenase-2 (COX-2), without affecting COX-2 mRNA. Lithium also reduced the brain concentration of prostaglandin E(2) (PGE(2)), a bioactive product of AA formed via the COX reaction. COX-1 and the Ca(2+)-independent iPLA(2) (type VI) were unaffected by lithium. These and prior results indicate that lithium targets a part of the AA cascade that involves cPLA(2) and COX-2. This effect may contribute to lithium's therapeutic action in bipolar disorder.

Energy consumption by phospholipid metabolism in mammalian brain.

Until recently, brain phospholipid metabolism was thought to consume only 2% of the ATP consumed by the mammalian brain as a whole. In this paper, however, we calculate that 1.4% of total brain ATP consumption is consumed for the de novo synthesis of ether phospholipids and that another 5% is allocated to the phosphatidylinositide cycle. When added to previous estimates that fatty acid recycling within brain phospholipids and maintenance of membrane lipid asymmetries of acidic phospholipids consume, respectively, 5% and 8% of net brain ATP consumption, it appears that phospholipid metabolism can consume up to 20% of net brain ATP consumption. This new estimate is consistent with recent evidence that phospholipids actively participate in brain signaling and membrane remodeling, among other processes.

Chronic nutritional deprivation of n-3 alpha-linolenic acid does not affect n-6 arachidonic acid recycling within brain phospholipids of awake rats.

Using an in vivo fatty acid model and operational equations, we reported that esterified and unesterified concentrations of docosahexaenoic acid (DHA, 22 : 6 n-3) were markedly reduced in brains of third-generation (F3) rats nutritionally deprived of alpha-linolenic acid (18 : 3 n-3), and that DHA turnover within phospholipids was reduced as well. The concentration of docosapentaenoic acid (DPA, 22 : 5 n-6), an arachidonic acid (AA, 20 : 4 n-6) elongation/desaturation product, was barely detectable in control rats but was elevated in the deprived rats. In the present study, we used the same in vivo model, involving the intravenous infusion of radiolabeled AA to demonstrate that concentrations of unesterified and esterified AA, and turnover of AA within phospholipids, were not altered in brains of awake F3-generation n-3-deficient rats, compared with control concentrations. Brain DPA-CoA could be measured in the deprived but not control rats, and AA-CoA was elevated in the deprived animals. These results indicated that AA and DHA are recycled within brain phospholipids independently of each other, suggesting that recycling is regulated independently by AA- and DHA-selective enzymes, respectively. Competition among n-3 and n-6 fatty acids within brain probably does not occur at the level of recycling, but at levels of elongation and desaturation (hence greater production of DPA during n-3 deprivation), or conversion to bioactive eicosanoids and other metabolites.

Chronic valproate treatment decreases the in vivo turnover of arachidonic acid in brain phospholipids: a possible common effect of mood stabilizers.

Both (Li(+)) and valproic acid (VPA) are effective in treating bipolar disorder, but the pathway by which either works, and whether it is common to both drugs, is not agreed upon. We recently reported, using an in vivo fatty acid model, that Li(+) reduces the turnover rate of the second messenger arachidonic acid (AA) by 80% in brain phospholipids of the awake rat, without changing turnover rates of docosahexaenoic or palmitic acid. Reduced AA turnover was accompanied by down-regulation of gene expression and protein levels of an AA-specific cytosolic phospholipase A(2) (cPLA(2)). To see if VPA had the same effect on AA turnover, we used our in vivo fatty acid model in rats chronically administered VPA (200 mg/kg, i.p. for 30 days). Like Li(+), VPA treatment significantly decreased AA turnover within brain phospholipids (by 28-33%), although it had no effect on cPLA(2) protein levels. Thus, both mood stabilizers, Li(+) and VPA have a common action in reducing AA turnover in brain phospholipids, albeit by different mechanisms.

Intravenously injected [1-14C]arachidonic acid targets phospholipids, and [1-14C]palmitic acid targets neutral lipids in hearts of awake rats.

The differential uptake and targeting of intravenously infused [1-14C]palmitic ([1-14C]16:0) and [1-14C]arachidonic ([1-14C]20:4n-6) acids into heart lipid pools were determined in awake adult male rats. The fatty acid tracers were infused (170 microCi/kg) through the femoral vein at a constant rate of 0.4 mL/min over 5 min. At 10 min postinfusion, the rats were killed using pentobarbital. The hearts were rapidly removed, washed free of exogenous blood, and frozen in dry ice. Arterial blood was withdrawn over the course of the experiment to determine plasma radiotracer levels. Lipids were extracted from heart tissue using a two-phase system, and total radioactivity was measured in the nonvolatile aqueous and organic fractions. Both fatty acid tracers had similar plasma curves, but were differentially distributed into heart lipid compartments. The extent of [1-14C]20:4n-6 esterification into heart phospholipids, primarily choline glycerophospholipids, was elevated 3.5-fold compared to [1-14C]16:0. The unilateral incorporation coefficient, k*, which represents tissue radioactivity divided by the integrated plasma radioactivity for heart phospholipid, was sevenfold greater for [1-14C]20:4n-6 than for [1-14C]16:0. In contrast, [1-14C]16:0 was esterified mainly into heart neutral lipids, primarily triacylglycerols (TG), and was also found in the nonvolatile aqueous compartment. Thus, in rat heart, [1-14C]20:4n-6 was primarily targeted for esterification into phospholipids, while [1-14C]16:0 was targeted for esterification into TG or metabolized into nonvolatile aqueous components.

Effects of single dose, postinduction dexamethasone on recovery after cardiac surgery.

BACKGROUND: Corticosteroids have been recommended to facilitate rapid recovery after cardiac surgery. We previously reported that dexamethasone given after induction of anesthesia decreases the incidence of postoperative shivering. We performed a post hoc analysis of the data obtained during that study, focusing on secondary outcomes. METHODS: A total of 235 adult patients undergoing elective coronary or valvular heart surgery were randomized to receive dexamethasone 0.6 mg/kg or placebo after induction of anesthesia. Patients who had pharmacologically treated diabetes mellitus, had hypersensitivity to dexamethasone, or were receiving treatment with corticosteroids were excluded. RESULTS: We found that, compared with placebo, patients receiving dexamethasone were more likely to remain tracheally intubated for 6 hours or less (26.4% vs 10.0%, p = 0.020) and had a lower incidence of early postoperative fever (20.2% vs 36.8%, p = 0.009) and new-onset atrial fibrillation during the first 3 days postoperatively (18.9% vs 32.3%, p = 0.027). However, we could not demonstrate a statistical difference in the intensive care unit or hospital length of stay, or in overall morbidity and mortality. The dexamethasone-treated patients were also more likely to have a higher blood glucose on admission to the intensive care unit (186 mg/dL vs 143 mg/dL, p = 0.012). CONCLUSIONS: Dexamethasone facilitates early tracheal extubation and is associated with a lower incidence of early postoperative fever and new-onset atrial fibrillation. Apart from a treatable decreased glucose tolerance, dexamethasone treatment was not shown to affect morbidity or mortality significantly.

85 kDa cytosolic phospholipase A2 is a target for chronic lithium in rat brain.

The mechanism by which chronic lithium exerts its therapeutic effect in brains of bipolar patients is not known. One possibility, suggested by our demonstration in the rat brain, is that chronic lithium inhibits turnover of arachidonic acid (AA) by reducing the activity of an AA-specific phospholipase A2 (PLA2). To test this further, mRNA levels of two AA-specific PLA2s, cytosolic PLA2 (cPLA2) type IV and intracellular PLA2 (iPLA2) type VIII, and protein level of cPLA2 were quantified in the brain of rats given lithium for 6 weeks. Chronic lithium markedly reduced brain mRNA and protein level of cPLA2, but had no effect on mRNA level of iPLA2. These results suggest that the final common path effect of chronic lithium administration is to reduce turnover of AA in brain by down-regulating cPLA2.

Effects of maturation on the phospholipid and phospholipid fatty acid compositions in primary rat cortical astrocyte cell cultures.

Phospholipid and phospholipid fatty acid compositional changes were studied in rat cortical astrocytes during dibutyryl cyclic adenosine monophosphate (dBcAMP, 0.25 mM) treatment starting after 14 days in culture (DIC). After 15 DIC, ethanolamine- and choline glycerophospholipid levels were increased 1.2- and 1.3-fold, respectively in treated compared to control cells. However, after 21 and 28 DIC, these levels were not significantly different between groups. Both groups had an increase in phosphatidylserine levels with increasing time in culture. Similarly, ethanolamine plasmalogen levels were transiently elevated after 21 DIC, but returned to previous levels after 28 DIC. The phospholipid fatty acid compositions for the acid stable and labile ethanolamine- and choline glycerophospholipids indicated that in dBcAMP treated cells, 20:4 n-6 and 22:6 n-3 proportions were elevated with increasing time in culture relative to control cells. As 20:4 n-6 proportions increased, there was a concomitant decrease in 20:3 n-9 proportions, suggesting an up regulation of n-6 series elongation and desaturation. In contrast, in control cells, the 20:4 n-6 proportions decreased with a corresponding increase in the 20:3 n-9 proportions. Thus, in treated cells, the cellular phospholipid fatty acid composition was dramatically different than control cells, suggesting that dBcAMP treatment may act to increase fatty acid elongation and desaturation.

Phospholipase A2 and its role in brain tissue.

Phospholipase A2 (PLA2) is the name for the class of lipolytic enzymes that hydrolyze the acyl group from the sn-2 position of glycerophospholipids, generating free fatty acids and lysophospholipids. The products of the PLA2-catalyzed reaction can potentially act as second messengers themselves, or be further metabolized to eicosanoids, platelet-activating factor, and lysophosphatidic acid. All of these are recognized as bioactive lipids that can potentially alter many ongoing cellular processes. The presence of PLA2 in the central nervous system, accompanied by the relatively large quantity of potential substrate, poses an interesting dilemma as to the role PLA2 has during both physiologic and pathologic states. Several different PLA2 enzymes exist in brain, some of which have been partially characterized. They are classified into two subtypes, Ca2+-dependent and Ca2+-independent, based on their catalytic dependence on Ca2+. Under physiologic conditions, PLA2 may be involved in phospholipid turnover, membrane remodeling, exocytosis, detoxification of phospholipid peroxides, and neurotransmitter release. However, under pathological situations, increased PLA2 activity may result in the loss of essential membrane glycerophospholipids, resulting in altered membrane permeability, ion homeostasis, increased free fatty acid release, and the accumulation of lipid peroxides. These processes, along with loss of ATP, may be responsible for the loss of membrane phospholipid and subsequent neuronal injury found in ischemia, spinal cord injury, and other neurodegenerative diseases. This review outlines the current knowledge of the PLA2 found in the central nervous system and attempts to define the role of PLA2 during both physiologic and pathologic conditions.

Separation of neutral lipids by high-performance liquid chromatography: quantification by ultraviolet, light scattering and fluorescence detection.

The recent increased use of cell cultures to model physiological events, in particular signal transduction and traumatic injury, has produced a need for a quantitative, high-performance liquid chromatographic separation of neutral lipid classes with a high degree of resolution and reproducibility. We report an isocratic separation using a Phenomenex Selectosil silica column (5 microns). Two solvents were used, 1.2% 2-propanol in n-hexane containing 0.1% acetic acid (90%) and n-hexane (10%) at a flow-rate of 0.6 ml/min. Column temperature was maintained at 55 degrees C and this temperature was critical for baseline resolution of 1,3-diacylglycerol and cholesterol. The use of 10% n-hexane permitted the resolution of low polarity compounds such as butylated hydroxytoluene, triacylglycerols and cholesteryl esters. All of the detectors used produced standard curves with linearity over a wide concentration range.

Altered lipid metabolism in the presence and absence of extracellular Ca2+ during combined oxygen-glucose deprivation in primary astrocyte cultures.

The effect of combined oxygen-glucose deprivation (COGD) on lipid metabolism in primary rat cortical astrocyte cultures was studied in both the presence and absence of extracellular Ca2+. In this study, increases in intracellular Ca2+ from internal Ca2+ stores were not inhibited nor were internal Ca2+ levels buffered. Combined oxygen-glucose deprivation resulted in a quantitative reduction in phospholipid levels and an increase in free fatty acid and lysophospholipid levels. Four hours after the onset of COGD, ethanolamine- and choline glycerophospholipid levels were decreased by 40 and 46% from control levels in the presence of Ca2+, respectively. A similar decrease was found 6 hr after onset of COGD in the absence of Ca2+. These changes were accompanied by elevated levels of the corresponding lysophospholipids. However, the increases in lysophospholipid content did not account for the entire loss of ethanolamine- or choline glycerophospholipid. Phosphatidylserine was reduced in both the presence and absence of extracellular Ca2+ but phosphatidylinositol was only decreased in the absence of Ca2+. Statistically significant increases in total fatty acid (FA) and polyunsaturated fatty acid (PUFA) levels occurred at 30 min and 3 hr after the onset of COGD in the absence and presence of Ca2+, respectively. Arachidonic acid levels were increased in both groups by 1 hr. These increases in FA, PUFA, and specifically arachidonic acid were time-dependent and increased over the 12 hr of COGD. Collectively, these results indicate the activation of an acylhydrolase mechanism in the possible presence of an inhibited reacylation pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Tritium isotope effect in high-performance liquid chromatography of eicosanoids.

A significant difference in retention time between unlabeled and the corresponding multi-tritium-labeled eicosanoid has been observed in the high performance liquid chromatography (HPLC) analysis of 11 eicosanoids. Variations in retention time range from 3-7%, depending on the separation conditions as well as the number and position of the tritium substitution. Multi-tritium-labeled eicosanoids were eluted earlier than the corresponding unlabeled eicosanoid in reversed phase HPLC, whereas no isotope effect was seen with 14C- and 3H2-eicosanoids. Considerations must be given to this tritium isotope effect whenever both multi-tritium-labeled and unlabeled eicosanoids are used for HPLC cochromatography or recovery studies.

High-performance liquid chromatography separation and quantitation of methylprednisolone from rat brain.

A sensitive, reliable method for the extraction, separation, and quantitation of methylprednisolone from rat brain is reported. The method can accurately quantitate methylprednisolone levels between 9.8 and 2500 ng/injection using a two-step HPLC separation and monitoring absorbance at 254 nm. A 90% extraction recovery of methylprednisolone (interday variation of 9.0% and an intraday variation of 0.0 to 7.7%) from rat cortex was obtained with a double extraction method using low toxicity solvents. These solvents are known to quantitatively extract the neutral lipids and phospholipids from brain. Combined with the ability to separate the neutral lipid and methylprednisolone fractions for further separation, and the ability to separate all phospholipid classes in the first run, this method offers great utility combined with the reliable, high extraction recovery and sensitive quantitation of methylprednisolone.

Oxygen-detoxifying enzymes in neutrophils of infants and their mothers.

The oxygen-detoxifying enzymes of neutrophils, SOD, GPX, and catalase, were measured in neutrophils obtained from normal human infants, their mothers, and controls to determine whether or not the impaired functions of infant neutrophils might be related to a decreased ability of these cells to detoxify reactive forms of oxygen. The rationale was based on the following. (1) Defective functions have been reported in neutrophils sustaining oxidative damage. (2) Increased oxidative metabolism and decreased functions can be demonstrated concomitantly in infant neutrophils. (3) Neutrophils from infants may be analogous to infant erythrocytes, cells known to exhibit increased susceptibility to oxidative injury and dysfunctions that are apparently related to deficiencies of GPX and catalase. SOD activity was similar in neutrophils obtained from infants, their mothers, and controls, whereas both GPX and catalase were significantly decreased in infant cells. The data suggest that infant neutrophils were rendered susceptible to oxidative damage and possibly to defective function by an imbalance of oxygen-detoxifying enzymes.

Neutrophil chemiluminescence during the first month of life.

Postphagocytic chemiluminescence of neutrophils was evaluated in infants throughout the 1st month of life, in their mothers and healthy adults to investigate further the defective chemiluminescence of neonatal neutrophils described by earlier studies. Maternal and adult values were similar and served as controls. Abnormalities both of peak chemiluminescence and of the kinetics of light emission were detected in infant neutrophils throughout the 1st month of life. Infant responses were variable, particularly at birth, when neutrophils of some infants performed comparably to those of controls.