Evaluation of the i-STAT (portable clinical analyser) for measuring haematological parameters in Atlantic cod (Gadus morhua) at different CO2 and temperature conditions.

Portable clinical analysers are gradually being involved in on-site assessment of haematic parameters in fish. The purpose of this study was to evaluate the i-STAT portable clinical analyser (i-STAT PCA) for accuracy and reliability of measuring blood pH, partial pressure of oxygen (pO2), haematocrit, haemoglobin, sodium, potassium and calcium in Atlantic cod (Gadus morhua). Haematological parameters detected with the i-STAT PCA were compared with conventional laboratory techniques (CLTs). Two types of disposable cartridges were used (CHEM8+ and CG4+) with the i-STAT PCA, and experiments were performed at two different temperature regimes (5 °C and 15 °C) and four different carbon dioxide (CO2) levels (0%, 0.1%, 0.5% and 1%). All blood parameters measured with the i-STAT PCA showed heterogeneous inaccuracy under the tested conditions, but the highest discrepancies were registered in blood pO2. The i-STAT PCA systematically overestimated the pO2 measurements. Our research suggests that i-STAT PCA is not an appropriate tool for pO2 measurements especially in coldwater fish species. The i-STAT PCA consistently underestimated the pH and haematocrit values especially at a lower temperature, although those parameters indicate significant high correlation at 15 °C. Furthermore, the analysed ions showed overestimation of sodium and underestimation of potassium and calcium.

Screening for protein adducts of naphthalene and chrysene in plasma of exposed Atlantic cod (Gadus morhua).

Polycyclic aromatic hydrocarbons (PAHs) are well known contaminants, ubiquitously present in the habitat and spawning areas for Atlantic cod (Gadus morhua). The Atlantic cod is a key species and a globally important food source, thus continuous monitoring of PAHs is considered highly valuable to ensure ecosystem sustainability and human food safety. PAH adducts to plasma proteins are applied as sensitive biomarkers of PAH exposure in humans and other species, thus the presence of PAH protein adducts in Atlantic cod plasma was investigated to identify PAH protein adduct biomarker candidates of exposure to PAHs. Blood plasma samples were collected from Atlantic cod (n = 66) one week after exposure by intramuscular injection of single PAHs (i.e. naphthalene and chrysene), and their corresponding dihydrodiol metabolites (i.e. (-)-(1R,2R)-1,2-dihydronaphthalene-1,2-diol and (-)-(1R,2R)-1,2-dihydrochrysene-1,2-diol). The samples were analyzed by shotgun tandem mass spectrometry (MS) and the resulting MS data were analyzed in Byonic™ to screen for proteins susceptible to adduct formation with naphthalene and chrysene. Furthermore, a wildcard modification search was performed to obtain additional information regarding potential modifications other than the targeted metabolites. The amino acid adductation sites and the metabolites involved in PAH adductation are reported. Forty-four proteins were found to bind PAHs. Alpha-2-macroglobulin-like proteins, apolipoproteins B-100-like proteins and an alpha-2-HS-glycoprotein were detected with the highest number of bound PAHs. This first insight into PAH protein adducts of Atlantic cod plasma generates valuable knowledge for the development of highly sensitive biomarkers of PAH exposure.

Effects of dietary arachidonic acid on the reproductive physiology of female Atlantic cod (Gadus morhua L.).

The present study was designed to investigate potential effects of arachidonic acid (ARA) on the reproductive physiology of female Atlantic cod (Gadus morhua L.). Two-year old Atlantic cod of both sexes were equally distributed into eight sea cages after completion of their first spawning in May 2005. Four experimental groups were established and fed diets with different levels of ARA corresponding to 0.5, 1, 2 and 4% of total fatty acid. Ovarian growth and development was documented every month. Fatty acid composition was analysed in ovaries, liver and plasma at the beginning of the experiment, one month prior to spawning, and in spent fish, one month after spawning was completed. Plasma concentrations of estradiol-17ß, testosterone and vitellogenin, and ovarian gene transcript levels of steroidogenic acute regulatory protein (star), P450aromatase (cyp19a1a) and 20ß-hydroxy steroid dehydrogenase (20bhsd/cbr1) were monitored every month in fish fed the experimental diets and related to oocyte stage. Potential fecundity was calculated based on ovarian samples taken one month before onset of spawning. Ovarian and plasma ARA levels were highly correlated to dietary ARA levels. There was a net accumulation of ARA compared to other essential fatty acids in ovarian tissue that was reflected in a decrease in EPA:ARA ratio. Plasma concentrations of vitellogenin, estradiol-17ß and testosterone and key gene transcript levels were affected by dietary ARA and stage of maturation. The results show that ARA has a significant influence on the reproductive physiology of female Atlantic cod.

Study of the plasma proteome of Atlantic cod (Gadus morhua): Effect of exposure to two PAHs and their corresponding diols.

Occurrence of polycyclic aromatic hydrocarbon (PAH) contamination in the marine environment represents a risk to marine life and humans. In this study, plasma samples from Atlantic cod (Gadus morhua) were analysed by shotgun mass spectrometry to investigate the plasma proteome in response to exposure to single PAHs (naphthalene or chrysene) and their corresponding metabolites (dihydrodiols). In total, 369 proteins were identified and ranked according to their relative abundance. The levels of 12 proteins were found significantly altered in PAH exposed fish and are proposed as new biomarker candidates. Eleven proteins were upregulated, primarily immunoglobulin components, and one protein was downregulated (antifreeze protein type IV.) The uniformity of the upregulated proteins suggests a triggered immune response in the exposed fish. Overall, the results provide valuable knowledge for future studies of the Atlantic cod plasma proteome and generate grounds for establishing new plasma protein biomarkers for environmental monitoring of PAH related exposure.

Life on the edge: O2 binding in Atlantic cod red blood cells near their southern distribution limit is not sensitive to temperature or haemoglobin genotype.

Atlantic cod are a commercially important species believed to be threatened by warming seas near their southern, equatorward upper thermal edge of distribution. Limitations to circulatory O2 transport, in particular cardiac output, and the geographic distribution of functionally different haemoglobin (Hb) genotypes have separately been suggested to play a role in setting thermal tolerance in this species. The present study assessed the thermal sensitivity of O2 binding in Atlantic cod red blood cells with different Hb genotypes near their upper thermal distribution limit and modelled its consequences for the arterio-venous O2 saturation difference, Sa-vO2 , another major determinant of circulatory O2 supply rate. The results showed statistically indistinguishable red blood cell O2 binding between the three HbI genotypes in wild-caught Atlantic cod from the Irish Sea (53° N). Red blood cells had an unusually low O2 affinity, with reduced or even reversed thermal sensitivity between pH 7.4 and 7.9, and 5.0 and 20.0°C. This was paired with strongly pH-dependent affinity and cooperativity of red blood cell O2 binding (Bohr and Root effects). Modelling of Sa-vO2  at physiological pH, temperature and O2 partial pressures revealed a substantial capacity for increases in Sa-vO2  to meet rising tissue O2 demands at 5.0 and 12.5°C, but not at 20°C. Furthermore, there was no evidence for an increase of maximal Sa-vO2  with temperature. It is suggested that Atlantic cod at such high temperatures may solely depend on increases in cardiac output and blood O2 capacity, or thermal acclimatisation of metabolic rate, for matching circulatory O2 supply to tissue demand.

Effects of amino acid supplementations on metabolic and physiological parameters in Atlantic cod (Gadus morhua) under stress.

The effects of tryptophan (Trp) and phenylalanine (Phe) diet supplementation on the stress and metabolism of the Atlantic cod have been studied. Fish were fed diet supplemented with Trp or Phe or control diet for 1 week. At the end of the feeding trial, fish were subjected to air exposure or heat shock. Following samples of blood, liver and muscle were taken from the fish and were analyzed for stress and metabolic indicators. After an air exposure, plasma cortisol levels in fish fed with Trp and Phe diets were lower compared to the fish fed the control diet. Diets containing both amino acids increased significantly the liver transaminase activities in juvenile cod. During thermal stress, high Trp contents had significant effects on fructose biphosphatase activity though Phe did not. Overall, activities of glucose 6-phosphate dehydrogenase, pyruvate kinase, and phosphofructokinase increased significantly for both amino acid diets. For the thermal stress, fish had the highest values of those activities for the 3Trp diet. Trp content in the diet had significant effects on the transaminase activity in muscle during air stress compared to fish fed control and Phe diets. Muscle alanine transaminase activity for thermal stress in fish fed any diet was not significantly different from the control. Both Trp and Phe supplementations reduced the stress markers in the cod; hence, they could be used as additives for the stress attenuation. However, they also raised the activity of key enzymes in glycolysis and gluconeogenesis, mainly the Trp diets.

Extracellular glucose supports lactate production but not aerobic metabolism in cardiomyocytes from both normoglycemic Atlantic cod and low glycemic short-horned sculpin.

Fish exhibit a wide range of species-specific blood glucose levels. How this relates to glucose utilization is yet to be fully realized. Here, we assessed glucose transport and metabolism in myocytes isolated from Atlantic cod (Gadus morhua) and short-horned sculpin (Myoxocephalus scorpius), species with blood glucose levels of 3.7 and 0.57 mmol l(-1), respectively. Glucose metabolism was assessed by the production of (3)H2O from [2-(3)H]glucose. Glucose metabolism was 3.5- to 6-fold higher by myocytes from Atlantic cod than by those from short-horned sculpin at the same level of extracellular glucose. In Atlantic cod myocytes, glucose metabolism displayed what appears to be a saturable component with respect to extracellular glucose, and cytochalasin B inhibited glucose metabolism. These features revealed a facilitated glucose diffusion mechanism that accounts for between 30% and 55% of glucose entry at physiological levels of extracellular glucose. Facilitated glucose diffusion appears to be minimal in myocytes for short-horned sculpin. Glucose entry by simple diffusion occurs in both cell types with the same linear relationship between glucose metabolism and extracellular glucose concentration, presumably due to similarities in membrane composition. Oxygen consumption by myocytes incubated in medium containing physiological levels of extracellular glucose (Atlantic cod 5 mmol l(-1), short-horned sculpin 0.5 mmol l(-1)) was similar in the two species and was not decreased by cytochalasin B, suggesting that these cells have the capability of oxidizing alternative on-board metabolic fuels. Cells produced lactate at low rates but glycogen levels did not change during the incubation period. In cells from both species, glucose utilization assessed by both simple chemical analysis of glucose disappearance from the medium and (3)H2O production was half the rate of lactate production and as such extracellular glucose was not available for oxidative metabolism. Overall, extracellular glucose makes only a minor contribution to ATP production but a sustained glycolysis may be necessary to support Ca(2+) transport mechanisms at either the sarcoplasmic reticulum or the sarcolemmal membrane.

Ocean warming and acidification modulate energy budget and gill ion regulatory mechanisms in Atlantic cod (Gadus morhua).

Ocean warming and acidification are threatening marine ecosystems. In marine animals, acidification is thought to enhance ion regulatory costs and thereby baseline energy demand, while elevated temperature also increases baseline metabolic rate. Here we investigated standard metabolic rates (SMR) and plasma parameters of Atlantic cod (Gadus morhua) after 3-4 weeks of exposure to ambient and future PCO2 levels (550, 1200 and 2200 µatm) and at two temperatures (10, 18 °C). In vivo branchial ion regulatory costs were studied in isolated, perfused gill preparations. Animals reared at 18 °C responded to increasing CO2 by elevating SMR, in contrast to specimens at 10 °C. Isolated gills at 10 °C and elevated PCO2 (≥1200 µatm) displayed increased soft tissue mass, in parallel to increased gill oxygen demand, indicating an increased fraction of gill in whole animal energy budget. Altered gill size was not found at 18 °C, where a shift in the use of ion regulation mechanisms occurred towards enhanced Na(+)/H(+)-exchange and HCO3 (-) transport at high PCO2 (2200 µatm), paralleled by higher Na(+)/K(+)-ATPase activities. This shift did not affect total gill energy consumption leaving whole animal energy budget unaffected. Higher Na(+)/K(+)-ATPase activities in the warmth might have compensated for enhanced branchial permeability and led to reduced plasma Na(+) and/or Cl(-) concentrations and slightly lowered osmolalities seen at 18 °C and 550 or 2200 µatm PCO2 in vivo. Overall, the gill as a key ion regulation organ seems to be highly effective in supporting the resilience of cod to effects of ocean warming and acidification.

Environmental genotoxicity and cytotoxicity in flounder (Platichthys flesus), herring (Clupea harengus) and Atlantic cod (Gadus morhua) from chemical munitions dumping zones in the southern Baltic Sea.

The data on environmental genotoxicity and cytotoxicity levels as well as on genotoxicity risk in flounder (Platichthys flesus), herring (Clupea harengus) and cod (Gadus morhua) collected in 2010-2012 at 42 stations located in chemical munitions dumping areas of the southern Baltic Sea are presented. The frequency of micronuclei, nuclear buds and nucleoplasmic bridges in erythrocytes was used as genotoxicity endpoint and the induction of fragmented-apoptotic, bi-nucleated and 8-shaped erythrocytes as cytotoxicity endpoint. The most significantly increased geno-cytotoxicity levels were determined in fish collected near known chemical munitions dumpsites. Extremely high genotoxicity risk for flounder were identified at 21 out of 24 stations, for herring at 29 out of 31 and for cod at 5 out of 10 stations studied. The reference level of genotoxicity was not recorded at any of the stations revealing that in the sampling area fish were affected generally.

The blood chemical status of Atlantic cod Gadus morhua following capture by jig and demersal longline with differential hook removal methods.

Common haematological [haematocrit (Hct)], primary (serum cortisol) and secondary (serum glucose and plasma lactate) analytes were utilized to compare blood biochemical status of Gadus morhua captured rapidly by jig with that of G. morhua captured by commercial demersal longline. In general, the physiological status of G. morhua, despite blind hook times, was significantly more disrupted (pronounced haemo-concentration and significantly elevated concentrations of cortisol, glucose and lactate) following longline capture relative to capture by jig, while no differences were detected among longline-caught fish as a function of dehooking method (or concomitant extent of overt physical trauma). Blood profiles from the more stressed G. morhua, a possible function of more extended longline hook times, were similar to the most stressed values reported for this species. The results also demonstrate that, although acute blood biochemical status is an effective gauge of relative stress, it does not reflect physical injury status, which has been shown to exert a strong influence on delayed mortality in previous studies in this species. Thus, acute blood chemical status alone may not be the most complete predictor of mortality. Future studies should evaluate physiological repercussions from capture-handling against physical trauma during more extended post-release periods for this species.

Pre-anaesthetic metomidate sedation delays the stress response after caudal artery cannulation in Atlantic cod (Gadus morhua).

Recovery from caudal artery cannulation with and without pre-anaesthesia metomidate sedation was assessed in Atlantic cod (Gadus morhua). The levels of plasma cortisol, glucose, electrolytes and acid-base parameters were compared between sedated and unsedated cod and to those in uncannulated individuals, where the samples were obtained by sacrificial sampling (reference level). Metomidate sedation delayed the stress response, causing sedated cod plasma cortisol to return to the reference level more slowly [day 4 post surgery (PS)] than in unsedated cod (day 2 PS). Plasma glucose was elevated in both sedated and unsedated cod up to and including day 5 PS. Plasma K(+) was lower and pH was higher in cannulated cod than in the reference from 24 h PS until the end of experimentation, indicating a stress effect of sacrificial sampling on plasma K(+) and pH that was likely caused by an acute stress response. Metomidate sedation delayed the stress response following CA cannulation and should therefore not be used as a pre-anaesthetic sedation in Atlantic cod. The caudal artery cannulation can be a useful tool in obtaining repeated blood samples from Atlantic cod given an adequate recovery time, which was determined to be 6 days irrespective of pre-anaesthesia sedation status.

An estrogen-responsive plasma protein expression signature in Atlantic cod (Gadus morhua) revealed by SELDI-TOF MS.

Compound-specific protein expression signatures (PESs) can be revealed by proteomic techniques. The SELDI-TOF MS approach is advantageous due to its simplicity and high-throughput capacity, however, there are concerns regarding the reproducibility of this method. The aim of this study was to define an estrogen-responsive PES in plasma of Atlantic cod (Gadus morhua) using the SELDI-TOF MS technique. Protein expression analysis of male cod exposed to 17ß-estradiol (E2) showed that 27 plasma peaks were differentially expressed following exposure. The reproducibility of this result was evaluated by reanalyzing the samples six months later, and a significant change in expression was confirmed for 13 of the 27 peaks detected in the first analysis. The performance of the reproducible E2-responsive PES, constituting these 13 peaks, was then tested on samples from juvenile cod exposed to 4-nonylphenol, North Sea oil, or North Sea oil spiked with alkylphenols. Principal component analysis revealed that nonylphenol-exposed cod could be separated from unexposed cod based on the E2-responsive PES, indicating that the PES can be used to assess estrogenic exposure of both juvenile and adult specimens of cod. A targeted antibody-assisted SELDI-TOF MS approach was carried out in an attempt to identify the E2-responsive peaks. Results indicated that 2 peaks were fragments of the well-known biomarkers VTG and/or ZRP. In this study, the SELDI-TOF MS technology has shown its potential for defining compound-specific PESs in fish. Nevertheless, thorough validation of reproducibility, specificity and sensitivity of a PES is required before it can be applied in environmental monitoring.

SELDI-TOF MS analysis of alkylphenol exposed Atlantic cod with phenotypic variation in gonadosomatic index.

Proteomics is a new and promising approach to evaluate potential effects of pollution. In order to investigate if there is a direct link between the protein expression profiles obtained by the SELDI-TOF MS technology and effects observed at the organism level in fish, plasma samples from unexposed and 20 ppb alkylphenol exposed female Atlantic cod (Gadus morhua) with high phenotypic variation in gonadosomatic index (GSI) were analyzed by SELDI-TOF MS. Principle component analysis (PCA) showed that the major proteomic variation present in the dataset (i.e. 23.6%) could be significantly correlated to the individual variation in GSI, which indicates that SELDI-TOF MS data can reflect effects observed at higher levels of organization in fish. Further exploration of the other principal components revealed an additional proteomic pattern specific for the alkylphenol exposed females. Hence, this study supports the usefulness of SELDI-TOF MS as a proteomic tool in ecotoxicological research.

From SELDI-TOF MS to protein identification by on-chip elution.

SELDI-TOF MS has been demonstrated as a powerful tool for biomarker discovery. However, a major disadvantage of SELDI-TOF MS is the lack of direct identification of the discriminatory peaks discovered. We describe a novel experimental identification strategy where peptides/proteins captured to a weak cation exchange ProteinArray surface (CM10) are eluted, and thereafter identified by utilizing a sensitive LC-MS/MS (i.e. LTQ Orbitrap). A mixture of four known proteins was used to test the novel experimental approach described, and all four proteins were successfully identified. Additionally, a biomarker candidate previously discovered in plasma of Atlantic cod (Gadus morhua) by SELDI-TOF MS was identified. Thus, this study indicated that a combination of on-chip elution and a highly sensitive LC-MS/MS system can be an alternative approach to identify biomarker candidates discovered by use of SELDI-TOF MS.

Endocrine modulation in Atlantic cod (Gadus morhua L.) exposed to alkylphenols, polyaromatic hydrocarbons, produced water, and dispersed oil.

Effluent from oil production activities contains chemicals that are suspected of inducing endocrine disruption in fish. In this study, Atlantic cod (Gadus morhua L.) were exposed to mixtures of low- and medium-molecular-weight alkylphenols (AP) (methyl- to heptylphenol), polycyclic aromatic hydrocarbons (PAH), diluted produced water, and dispersed oil for 15 d in a flow-through exposure system. Condition index (CI), hepatosomatic index (HSI), gonadosomatic index (GSI), concentration of the estrogenic biomarker vitellogenin (Vtg), and modulation of the total sex steroid-binding capacity in plasma were determined to assess whether these mixtures were capable of interfering with endocrine-regulated physiological processes in Atlantic cod. No marked differences in plasma Vtg levels were found between control and exposed groups of either males or females, possibly due to high intergroup variances and low sample numbers. An apparent numerical increase in the number of male and female fish with high plasma Vtg levels was, however, observed in some exposure groups compared to control. This purported weak estrogenic effect was several orders of magnitude lower than that observed for potent estrogens and suggested that the levels of estrogen receptor (ER) agonists were low. Exposure of female fish to a mixture of dispersed oil and a mixture of AP, PAH, and dispersed oil led to upregulation of the plasma total sex steroid-binding capacity, indicating interference with the normal blood steroid transport. No significant effects were seen for CI, HSI, and GSI, suggesting that the endocrine-disrupting potential was not sufficient to elicit effects on general physiological conditions and gonad development during this short exposure period.

Repeated sampling of Atlantic cod (Gadus morhua) for monitoring of nondestructive parameters during exposure to a synthetic produced water.

The past decades of monitoring discharges from oil and gas industry have revealed that although there are indications of adverse effects in tissues of aquatic organisms, little is known about their temporal development. Furthermore, observations in wild-caught individuals have not been clearly reproduced in laboratory studies or caging studies, and vice versa, and the results are therefore not easily interpretable. There is clearly a need for exposure studies designed for monitoring the development of effect markers in individual fish over chronic periods to low contaminant levels. Through repetitive nondestructive sampling, the progression of effects may be monitored in individuals, significantly reducing the number of fish needed in exposure studies. A laboratory exposure study was designed to be able to monitor selected parameters in individual Atlantic cod (Gadus morhua). Passive integrated transponders in combination with visible implant elastomers were used to study individual fish during the exposure period (44 wk). Fish were measured (weight and length) and a blood sample was taken for analysis of hematocrit, DNA damage (micronucleus), and oxidative stress (total oxyradical scavenging capacity) at up to seven time points. There were no apparent adverse effects of treatments on the health of experimental fish, frequency of micronucleated erythrocytes, or oxidative stress in whole blood. It is possible that the time scale was not sufficient for development and detection of parameters included here or that red blood cells may not be a suitable matrix for the selected analyses. Future studies need to include other parameters in blood to investigate their sensitivity to low-concentration exposures.

Effects of produced water on reproductive parameters in prespawning Atlantic cod (Gadus morhua).

Produced water (PW) discharged from offshore oil industry activities contains substances that are known to contribute to a range of mechanisms of toxicity. In the present study selected reproductive biomarkers were studied in prespawning Atlantic cod (Gadus morhua) exposed to PW. The fish were exposed for 12 wk within a continuous flow-through system at realistic environmental near-field concentrations. Concentrations of polyaromatic hydrocarbon (PAH) and alkylphenol (AP) compounds were analyzed by gas chromatography with mass spectrometric detection measurement, as were PAH and AP metabolites in fish bile for verification of exposure conditions and presence of compounds in PW. A suite of reproductive biomarkers (vitellogenin, zona radiata protein, and plasma steroid concentrations) and histological alterations of the gonads were determined. Results showed that exposure to sufficiently high levels of PW produced an increase in vitellogenin levels in female fish compared to the control. Impaired oocyte development and reduced estrogen levels were also observed in PW-exposed female fish. In male fish testicular development was altered, showing a rise in amount of spermatogonia and primary spermatocytes and a reduction in quantity of mature sperm in the PW-exposed fish compared to control. Data indicate that sufficiently high levels of PW have the potential to adversely affect the reproductive fitness of cod.

Water column monitoring of the biological effects of produced water from the Ekofisk offshore oil installation from 2006 to 2009.

The Norwegian water column monitoring program investigates the biological effects of offshore oil and gas activities in Norwegian waters. In three separate surveys in 2006, 2008, and 2009, bioaccumulation and biomarker responses were measured in mussels (Mytilus edulis) and Atlantic cod (Gadus morhua) held in cages at known distances from the produced water (PW) discharge at the Ekofisk oil field. Identical monitoring studies performed in all three years have allowed the biological effects and bioaccumulation data to be compared, and in addition, enabled the potential environmental benefits of a PW treatment system (CTour), implemented in 2008, to be evaluated. The results of the 2009 survey showed that caged animals were exposed to low levels of PW components, with highest tissue concentrations in mussels located closest to the PW discharge. Mussels located approximately 1-2 km away demonstrated only background concentrations of target compounds. Concentrations of polycyclic aromatic hydrocarbons (PAH) and alkyl phenol (AP) metabolites in the bile of caged cod were elevated at stations 200-250 m from the discharge. There was also a signal of exposure relative to discharge for the biomarkers CYP1A in fish and micronuclei in mussels. All other fish and mussel biomarkers showed no significant exposure effects in 2009. The mussel bioaccumulation data in 2009 indicated a lower exposure to the PW effluent than seen previously in 2008 and 2006, resulting in an associated general improvement in the health of the caged mussels. This was due to the reduction in overall discharge of PW components (measured as oil in water) into the area in 2009 compared to previous years as a result of the improved PW treatment system.

pH-induced shift in hemoglobin spectra: a spectrophotometeric comparison of atlantic cod ( Gadus morhua ) and mammalian hemoglobin.

Due to a pH-sensitive effect in many fish hemoglobins (Hb), analytical errors may occur when mammalian Hb is used as a standard in quantitative spectrophotometric multicomponent analysis of fish blood. The aim of this work was to examine differences in the optical spectra of mammalian (human) and fish (farmed Atlantic cod) Hb subjected to pH 7.4 and 6.5. The absorption spectra of the common derivatives, deoxy- (HHb), oxy- (OHb), carboxy- (COHb), and methemoglobin (metHb), were determined in the spectral range of 450-700 nm. The metHb spectra of fish differed considerably from the corresponding human Hb spectra, whereas only minor differences in OHb, HHb, and COHb were found. Cod Hb was significantly (P < 0.05) influenced by a drop in pH compared to mammalian Hb. This resulted in deoxygenation of the Hb and increased autoxidation. For human Hb, a pH-independent isosbestic point in the spectra of OHb, HHb, and metHb at 523 nm was found. This isosbestic point was not found in the absorption spectra of cod Hb. In conclusion, spectra of cod metHb and human metHb behave differently. This must thus be taken into account in spectrophotometric multicomponent analysis. Ideally, Hb in muscle or blood should be determined by comparison to a standard made from the same species.

Changes in arterial PO2, physiological blood parameters and intracellular antioxidants in free-swimming Atlantic cod (Gadus morhua) exposed to varying levels of hyperoxia.

Free-swimming Atlantic cod (Gadus morhua) were exposed to water oxygen pressures (P(w)O2) ranging from 18.1 to 41.5 kPa and sampled for blood using an indwelling caudal artery cannula. Arterial blood oxygen pressure (P(a)O2) increased with increasing P(w)O2, from 12.0 kPa in normoxia (18.1 kPa) to 34.2 kPa in the highest hyperoxic level tested (41.5 kPa). Blood CO2 pressure and plasma bicarbonate concentration increased with P(w)O2, indicating reduced ventilation with increased P(w)O2. Plasma glucose, sodium and potassium were not affected by water oxygen level. Blood oxidative stress biomarkers, reduced glutathione, oxidized glutathione and the oxidative stress index (ratio between oxidized and total glutathione) differed intermittently between normoxia and hyperoxia. The oxidative stress index was higher in the blood of exposed compared to unexposed control cod. Together with elevated P(a)O2, these findings suggest increased production of reactive oxygen species and increased oxidative stress in Atlantic cod exposed to hyperoxia.