A comparison of blood nitric oxide metabolites and hemoglobin functional properties among diving mammals.

The ability of marine mammals to hunt prey at depth is known to rely on enhanced oxygen stores and on selective distribution of blood flow, but the molecular mechanisms regulating blood flow and oxygen transport remain unresolved. To investigate the molecular mechanisms that may be important in regulating blood flow, we measured concentration of nitrite and S-nitrosothiols (SNO), two metabolites of the vasodilator nitric oxide (NO), in the blood of 5 species of marine mammals differing in their dive duration: bottlenose dolphin, South American sea lion, harbor seal, walrus and beluga whale. We also examined oxygen affinity, sensitivity to 2,3-diphosphoglycerate (DPG) and nitrite reductase activity of the hemoglobin (Hb) to search for possible adaptive variations in these functional properties. We found levels of plasma and red blood cells nitrite similar to those reported for terrestrial mammals, but unusually high concentrations of red blood cell SNO in bottlenose dolphin, walrus and beluga whale, suggesting enhanced SNO-dependent signaling in these species. Purified Hbs showed similar functional properties in terms of oxygen affinity and sensitivity to DPG, indicating that reported large variations in blood oxygen affinity among diving mammals likely derive from phenotypic variations in red blood cell DPG levels. The nitrite reductase activities of the Hbs were overall slightly higher than that of human Hb, with the Hb of beluga whale, capable of longest dives, having the highest activity. Taken together, these results underscore adaptive variations in circulatory NO metabolism in diving mammals but not in the oxygenation properties of the Hb.

Measurement of marine osmolytes in mammalian serum by liquid chromatography-tandem mass spectrometry.

Osmolytes are accumulated intracellularly to offset the effects of osmotic stress and protect cellular proteins against denaturation. Because different taxa accumulate different osmolytes, they can also be used as "dietary biomarkers" to study foraging. Potential osmolyte biomarkers include glycine betaine, trimethylamine N-oxide (TMAO), homarine, dimethylsulfoniopropionate (DMSP), and the osmolyte analog arsenobetaine (AsB). We present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous measurement of these osmolytes in serum or plasma. Varying concentrations of osmolytes were added to serum and samples and extracted in 90% acetonitrile and 10% methanol containing 10 µM deuterated internal standards (D(9)-glycine betaine, D(9)-trimethylamine-N-oxide, (13)C(2)-arsenobetaine, D(6)-DMSP, and D(4)-homarine). Analytes were separated on a normal-phase modified silica column and detected using isotope dilution tandem mass spectrometry in multiple reaction monitoring (MRM) mode. The assay was linear for all six compounds (r(2) values=0.983-0.996). Recoveries were greater than 85%, and precision for within-batch coefficients of variation (CVs) were less than 8.2% and between-batch CVs were less than 6.1%. Limits of detection ranged from 0.02 to 0.12 µmol/L. LC-MS/MS is a simple method with high throughput for measuring low levels of osmolytes that are often present in biological samples.

HAIR, WHOLE BLOOD, AND BLOOD-SOAKED CELLULOSE PAPER-BASED RISK ASSESSMENT OF MERCURY CONCENTRATIONS IN STRANDED CALIFORNIA PINNIPEDS.

Mercury (Hg) poses a health risk to wildlife populations and has been documented at relatively high concentrations in many marine mammals, including wild-caught pinnipeds along the central California, US coast. We measured total Hg concentrations ([THg]) in hair and blood of live-stranded harbor seals (HS; Phoca vitulina), California sea lions (CSL; Zalophus californianus), and northern elephant seals (NES; Mirounga angustirostris) in California to quantify species, temporal, and spatial variability in [THg] and assess the relationships between [THg] measured by different methods (blood vs. filter paper) and in different matrices (blood vs. hair). We compared [THg] with toxicologic thresholds of concern to aid in identification of at-risk individuals or groups and better understand how the use of different methods and matrices affects assumed toxicologic risk. There was a wide range of [THg] in blood (<0.01-1.13 µg/g) and hair (0.45-81.98 µg/g), and NES had higher [THg] compared with HS and CSL. All three species had individuals with [THg] that exceeded the lower threshold for one or both matrices, but only HS pups had [THg] exceeding upper thresholds. Spatial differences in [THg] were detected, with higher concentrations in HS pups from areas surrounding San Francisco Bay, but differences were dependent on sampling year and matrix. The relationship between [THg] in blood and filter paper (r2=0.98) was strong, and differences had little influence on comparisons with toxicologic thresholds. Blood and hair [THg] were generally in agreement (r2=0.72), but large mismatches for a few seals underscore the importance of combined sampling in adverse effects studies where accurate assessment of Hg exposure is crucial. The wide range of [THg] in stranded HS pups that exceeded published thresholds of concern makes them a promising candidate for adverse effects studies, particularly because different matrices represent Hg exposure across key developmental stages.

Absence of circadian patterns of secretion of melatonin or cortisol in Weddell seals under continuous natural daylight.

Blood samples were obtained from adult female Weddell seals in McMurdo Sound, Antarctica, utilizing four different seals every 2 h for 24 h on two occasions in November 1987. Plasma levels of melatonin were mostly near zero, whereas the values for cortisol were high (2101 +/- 54 and 1908 +/- 108 (S.E.M.) nmol/l, n = 52). For both hormones there was no evidence of a diurnal pattern of secretion. Juvenile seals sampled in December 1985 also had no evidence of a diurnal secretion pattern of melatonin, but blood levels of melatonin were higher than in adults. The results indicate that secretion of melatonin in adult Weddell seals ceases during the Antarctic summer.

Hemoglobin concentrations and blood gas tensions of free-diving Weddell seals.

Arterial blood gas tensions, pH, and hemoglobin concentrations were measured in four free-diving Weddell seals Leptonychotes weddelli. A microprocessor-controlled sampling system enabled us to obtain 24 single and 31 serial aortic blood samples. The arterial O2 tension (PaO2) at rest [78 +/- 13 (SD) Torr] increased with diving compression to a maximum measured value of 232 Torr and then rapidly decreased to 25-35 Torr. The lowest diving PaO2 we measured was 18 Torr just before the seal surfaced from a 27-min dive. A consistent increase of arterial hemoglobin concentrations from 15.1 +/- 1.10 to 22.4 +/- 1.41 g/100 ml (dives less than 17 min) and to 25.4 +/- 0.79 g/100 ml (dives greater than 17 min) occurred during each dive. We suggest that an extension of the sympathetic outflow of the diving reflex possibly caused profound contraction of the Weddell seal's very large spleen (0.89% of body wt at autopsy), although we have no direct evidence. This contraction may have injected large quantities of red blood cells (2/3 of the total) into the seal's central circulation during diving and allowed arterial O2 content to remain constant for the first 15-18 min of long dives. The increase of arterial CO2 tensions during the dive and the compression increase of arterial N2 tensions were also moderated by injecting red blood cells sequestered at ambient pressure. After each dive circulating red blood cells are oxygenated and rapidly sequestered, possibly in the spleen during the first 15 min of recovery.

Microcomputer monitor and blood sampler for free-diving Weddell seals.

The equipment used for the first sampling of arterial blood at depth on free-diving Weddell seals Leptonychotes weddelli is described. Blood was withdrawn through an aortic catheter by a submersible, peristaltic roller pump and stored in a single- or multiple-sample collection device. The multiple sampler allowed up to eight individual blood samples to be collected during a single dive. The blood pump was controlled by a dedicated microcomputer that allowed initiation of blood sampling at flexible combinations of depth and/or time during either the descending or ascending phase of the dive. The dedicated microcomputer also recorded swimming depth, velocity, heart rate, and body temperature at selectable time intervals. These data were transmitted to a laboratory computer, and blood samples were retrieved, when the seal surfaced to breathe.

Characterization and measurement of immunoglobulins in the grey seal (Halichoerus grypus).

Immunoglobulin concentrations were measured in the serum and colostrum of adult and pup Grey seals from North Rona in the outer Hebrides and the Isle of May in the Firth of Forth. IgG was shown to be the main immunoglobulin and two subclasses were identified. Serum immunoglobulin content was low in the week-old pups and increased up to 5 weeks, although it remained substantially lower than in the adults. Colostral immunoglobulins were high and cannot explain the low values in the pup serum. It is considered that the increased opportunistic infections seen in Grey seal pups may be related to this low immunoglobulin status.

Red cell aggregation and viscoelasticity of blood from seals, swine and man.

RBC aggregation and viscoelasticity parameters were determined for 40% suspensions of washed cells in autologous plasma from elephant seals (ES), Mirounga angustirostris, ringed seals (RS), Phoca hispida, and swine, (SS), Sus scrofa. Interspecific comparisons including human (HS) blood data revealed unusual rheological properties of seal blood relative to that from pigs or man: 1) RBC aggregation extent, rate and sedimentation were lower for seals (AI = 0, ZSR = .40, ESR = 0 for RS blood) relative to humans; 2) Viscous (n') and elastic (n") components of complex viscosity (OCRD) were lower for both seal species relative to SS blood, but only at shear rates less than or equal to 10 sec-1 (P less than 0.05), while n"/n' ratios for RS blood were lower than HS blood at all shear rates (P less than 0.01); 3) Blood viscosity measurements for RS and SS blood from rotational viscometry (Contraves) were consistent with OCRD data; 4) Seal plasma fibrinogen levels were low compared to pigs or humans (RS fibrinogen = -43% v. HS and -57% v. SS; ES fibrinogen = -58% v. HS and -69% v. SS). Electrophoretic mobility of RS red cells was +25% relative to those of humans. These results demonstrate differences in hemorheological indices among mammalian species and suggest the value of comparative rheologic studies.

Haematology of the Australian sea lion, Neophoca cinerea.

The haematology of the Australian sea lion Neophoca cinerea was studied in a breeding colony on Kangaroo Island, South Australia. The methods used to catch and restrain the animals are described and the haematology of 38 animals of varying age and sex groups is recorded. Total values for both erythrocytes and leucocytes were similar to those of other marine mammals and were in the ranges of 4.77 to 6.08 X 10(6) mm3 and 6.3 to 14.6 X 10(3) mm3, respectively. Erythrocytes volumes were very large, measuring from 96 to 112 mm.3 The packed cell volumes ranged from 48.3 to 64.2% and the haemoglobin values from 16.2 to 21 gm percent. The neutrophil lymphocyte ratio varied from 0.5 to 6.2 and in some animals absolute lymphocyte values were less than 1,200 mm.3 In many animals the percentage of eosinophils was greater than 20%, suggesting parasitic disease.

Clinical blood values of the northern fur seal, Callorhinus ursinus. II. Comparison of fresh versus stored frozen serum.

Analysis of 14 blood components in fresh and stored (107-166 days) frozen serum of the northern fur seal (Callorhinus ursinus) revealed significant changes in the values of all but total protein, globulins, phosphorus and creatinine. While most values decreased during storage, cholesterol and bilirubin showed small but significant increases.

Hematology and serum chemistry of the young Hawaiian monk seal (Monachus schauinslandi).

Between January 1984 and May 1987, blood samples were collected from 12 young (3- to 6-mo-old) Hawaiian monk seals (Monachus schauinslandi) that were captured in the wild and held in captivity. All samples evaluated were from clinically normal animals. Average hematologic and serum chemistry values were not remarkable for a young diving mammal. The blood and serum analyses performed established reference ranges, which can be used as indicators of health status for this endangered species.

Plasma biochemical values of clinically-normal Australian sea lions (Neophoca cinerea).

Seventeen biochemical constituents were assayed in the blood plasma of clinically-normal Australian sea lions (Neophoca cinerea). The sea lions formed part of a breeding colony which inhabits the southern coast of Kangaroo Island, South Australia. Little variation was found in any of the values obtained from animals of different age and sex. The results were compared with values published for California sea lions (Zalophus californianus), Northern fur seals (Callorhinus ursinus), Harbor seals (Phoca vitulina) and Northern elephant seals (Mirounga angustirostris).

Effects of handling stress on plasma enzymes in harp seals, Phoca groenlandica.

Three harp seal pups, Phoca groenlandica, were captured on the ice of the Gulf of St. Lawrence, and subjected to 3 h of transportation and handling stress. The activities of creatine kinase (CK), aspartate aminotransferase (AspAT), aldolase, alanine aminotransferase, gamma glutamyl transpeptidase, and leucine aminopeptidase were determined in serial blood samples collected for 4 d following the stress episode. Marked elevation of plasma CK activity was observed 3 h after capture. Values returned to normal in 12 h in two seals, and by 24 h in the third. Slight elevations in AspAT were also noted; the remaining enzymes were unaffected. Plasma CK is recommended as a sensitive indicator of handling stress in seals.

[Physiological and biochemical characteristics of the blood of the Greenland seal, Pagophilus groenlandicus]. / Fiziologicheskaia i biokhimicheskaia kharakteristiki krovi grenlandskogo tiulenia Pagophilus groenlandicus.

High hemoglobin concentration, rich hemoglobin supply of the body and high specific weight of the blood were found in harp seal cubs. The erythrocyte number is relatively low which agrees with our previous studies on aquatic and semi-aquatic mammals. Serum proteins from both adult and young harp seals are separated into 5-8 fractions during agar-agar gel electrophoresis. Diving of seals for considerable periods is partly accounted for by high hemoglobin and myoglobin concentrations adn also by perfect supply of the organism by these pigments.

Halogenated phenolic contaminants in the blood of marine mammals from Japanese coastal waters.

Information on accumulation of halogenated phenolic contaminants in the blood of marine mammal is limited. The present study, we determined the residue levels and patterns of chlorinated and brominated phenolic contaminants (OH-PCBs, OH-PBDEs and bromophenols) in the blood collected from pinnipeds (northern fur seal, spotted seal, Steller sea lion and ribbon seal) and small cetaceans (harbor porpoise and Dall's porpoise) from Japanese coastal waters. Concentrations of PCBs and OH-PCBs found in pinnipeds were the same as in small cetaceans living in the same coastal area. However, significantly lower concentrations of brominated compounds (PBDEs, MeO-PBDEs, OH-PBDEs) were found in the blood of pinnipeds than the levels found in cetacean species which live same area (p < 0.05). This difference of accumulation pattern suggested pinnipeds have an enhanced capability to degrade organobromine compounds relative to cetaceans.

Amino-acid sequence of the alpha and beta chains of adult hemoglobin of the harbor seal, Phoca vitulina.

The complete amino-acid sequences of the alpha and beta chains of adult hemoglobin of harbor seal, Phoca vitulina that belong to carnivora were determined as follows. The alpha and beta chains isolated by chromatography on a CM-cellulose column were digested with trypsin after S-carboxymethylation. Amino-acid sequences of the tryptic peptides derived from both chains were analysed. Comparing the primary structures of the alpha and beta chains of the seal hemoglobin with those of human, dog, bear, badger and cat, 19, 12, 12, 11, and 16 substitutions, respectively, were recognized in the alpha chain, and 12, 10, 4, 6, and 19 (22) in the beta chain.

Plasma concentrations of oestrone, progesterone and corticosteroids during late pregnancy and after parturition in the harbour seal, Phoca vitulina.

Blood samples were taken at weekly intervals during the last 2 months of pregnancy from a mature (7-year-old) harbour seal which had conceived and was kept in captivity. The concentrations of oestrone, oestrone sulphate, progesterone and glucocorticoids were determined by radioassays. The plasma levels of unconjugated oestrone were slightly greater than those of oestrone sulphate. Total oestrone declined steadily over the last month from a peak of 2.3 ng/ml at 30 days before parturition. Plasma progesterone concentrations rose to 61 ng/ml at 2 weeks before parturition and fell to about half that value by 2 days before birth of a normal male pup. Plasma glucocorticosteroids reached a peak of 164 ng/ml, also at 2 weeks, but showed only a slight decline thereafter. A higher value (392 ng/ml) was recorded 5 days after parturition.

Summer and winter cycles in plasma melatonin levels in the elephant seal (Mirounga leonina).

Plasma melatonin concentration of immature male elephant seals was determined by radioimmunoassay. Comparison of concentrations during two 24-h periods, one in midsummer and one in midwinter, showed that there was a marked circadian cycle in winter which was greatly modified during the long day length of summer. It is suggested that in summer there was sufficient ambient lighting during the night hours to depress the nocturnal rise in plasma melatonin. The complexity of pineal cycles in the natural environment is stressed, and in this regard the polar regions are of particular interest due to the extreme seasonal changes in day length there.

The lipid composition of serum lipoproteins in the harbour seal, Phoca vitulina.

The density profile of serum lipoproteins and their lipid composition was studied in 12 adult, female harbour seals. The animals were sampled after an approximate 20 hr fast. The density profile of lipoproteins showed that the harbour seals displayed a distinct VLDL (density less than 1.006 g/ml) and HDL band (density about 1.125 g/ml), but no clear LDL band. There was a rather diffuse population of lipoproteins in the density range of 1.019-1.100 g/ml. Mean serum total cholesterol concentration was 5.7 mmol/l; about 60% of this cholesterol was located in the HDL fraction (density greater than 1.063 g/ml). The fasted seals were found to carry 4% of serum total lipids in chylomicrons. These lipoproteins consisted of 51% of triaclyglycerols (on the basis of total chylomicron lipids). The LDL (defined as heparin-manganese precipitable lipoproteins in VLDL and chylomicron-deficient serum) contained 49% of cholesterol and 43% of phospholipids (on the basis of total LDL lipids). The HDL (defined as heparin-manganese soluble lipoproteins in VLDL and chylomicron-deficient serum) contained 36% of cholesterol and 58% of phospholipids (on the basis of total HDL lipids).