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.

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.

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.

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.

Temperature and the oxygen-hemoglobin dissociation curve of the harbor seal, Phoca vitulina.

To determine the effect of temperature and pH on oxygen-hemoglobin affinity of the harbor seal, we measured 61 biotonometric oxygen-hemoglobin dissociation curves on blood from 5 seals at 3 temperatures and a range of pH values. The average (+/- SEM) hemoglobin concentration was 3.44 +/- 0.15 mM, nearly 50% greater than found in normal humans. At pH 7.4 the P50 (partial pressure of O2 at 50% hemoglobin saturation) +/- SEM values were 22.4 +/- 0.6,25.3 +/- 0.5, and 28.5 +/- 0.4 Torr at 33, 37 and 41 degrees C, respectively. The effect of temperature on oxygen-hemoglobin affinity, (delta log P50/delta T) was 0.014 +/- 0.001 at pH 7.4, significantly lower than that observed in human and dog blood. This low temperature sensitivity may facilitate oxygen off-loading from hemoglobin when temperature gradients exist within the animal or as tissue temperature decreases during a dive. Temperature did not significantly affect the Hill coefficient 'n' (shape) of the dissociation curve which averaged 2.43 +/- 0.04 at 37 degrees C. The fixed-acid Bohr coefficient (delta log P50/delta pH) was -0.606 +/- 0.032 at 37 degrees C and increased with temperature. This relatively large value for the Bohr coefficient was similar to those previously reported for the Northern Elephant, Bladdernose, and Weddell Seals, and may facilitate oxygen off-loading as acidosis develops during a dive.

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.

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.

Arterial gas tensions and hemoglobin concentrations of the freely diving Weddell seal.

Heart rate, body temperature, arterial blood gas tensions, pHa and hemoglobin concentrations were measured during and after free diving of the Weddell seal (Leptonychotes weddelli), beneath the fast ice of McMurdo Sound, Antarctica. To examine arterial nitrogen tensions (PaN2), 47 samples of arterial blood were obtained from 4 seals diving up to 23 min to depths of 230 m. Peak arterial nitrogen tensions between 2000 and 2500 mmHg were recorded at depths of 40-80 m during descent, indicating that the seal's lung collapses by 25-50 m. Then arterial blood nitrogen tensions slowly decreased to about 1500 mmHg near surfacing. Thus during diving alveolar collapse and redistribution of blood nitrogen allow the seal to avoid nitrogen narcosis and decompression sickness. The arterial PO2 (PaO2) at rest (78 +/- 13 mmHg, mean +/- SD) increased with diving compression to a maximum measured value of 232 mmHg and then rapidly decreased to 25-35 mmHg. The lowest diving PaO2 was 18 mmHg just before the seal surfaced from a 27-min dive. A consistent increase of arterial hemoglobin concentrations occurred during each dive. We suggest that an extension of the sympathetic outflow of the diving reflex caused profound contraction of the Weddell seal's spleen.

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.

Carnivora: the primary structure of Weddell Seal (Leptonychotes weddelli, Pinnipedia) hemoglobin.

The hemoglobin of Weddell Seal (Leptonychotes weddelli, Pinnipedia) comprises two components with identical beta-chains. The alpha-chains differ in positions 15 (Gly/Asp) and 57 (Ala/Thr). We present the primary structure of the chains which have been separated by reversed-phase high-performance liquid chromatography. The sequences have been determined by automatic Edman-degradation with the film-technique or the gas-phase method, using the native chains and the tryptic peptides of the oxidized chains. Compared to the corresponding human chains we found 22 substitutions in the alpha-chains and 14 in the beta-chains. In the alpha-chains exchanges involve one heme- and three alpha 1/beta 1-contacts. In the beta-chains one heme contact, one alpha 1/beta 1- and one alpha 1/beta 2-contacts are substituted. The sequences are compared to those of other Pinnipedia and Arctoidea hemoglobins.

Influence of hematocrit on whole blood glucose levels: new evidence from marine mammals.

The water content and glucose concentration in the whole blood of marine mammals were found to be correlated to red blood cell concentration. Because hematocrit (Hct) undergoes significant periodic shifts in these mammals during periods of apnea and/or diving, the measured values of whole blood glucose change due to alterations in Hct, independent of shifts in metabolite regulatory pathways. In contrast to humans, where red blood cell and plasma glucose concentrations are equivalent, in most other mammalian species red blood cell glucose concentration is much lower than that in plasma. Therefore, as Hct increases, the whole blood concentration of glucose must decrease in a linear manner, depending on the magnitude of the Hct change and the concentration of glucose in the blood cells. This study demonstrates that water and metabolite distribution must be considered when interpreting whole blood metabolite data. The results suggest that plasma or serum analysis of metabolites would avoid distribution problems. This is especially critical in species where Hct varies during the course of the experiment, as in exercising or diving mammals.

Carnivora: the primary structure of the Pacific Walrus (Odobenus rosmarus divergens, Pinnipedia) hemoglobin.

The primary structure of the alpha- and beta-chains of the hemoglobin from the Pacific Walrus (Odobenus rosmarus divergens, Pinnipedia) is presented. Sequence analysis revealed only one hemoglobin component whereas two bands were found in polyacrylamide gel electrophoresis. The globin chains were separated by high-performance liquid chromatography and the sequences determined by automatic liquid- and gas-phase sequencing of the chains and their tryptic peptides. The alpha-chains show 20 and the beta-chains 12 exchanges compared to the corresponding human chains. In the alpha-chains one heme- and two alpha 1/beta 1-contacts were exchanged whereas in the beta-chains one alpha 1/beta 1-, one alpha 1/beta 2-and one heme-contact are substituted. Compared to Harbour Seal (Phoca vitulina) the Walrus hemoglobin shows 9 amino-acid replacements in the alpha-chains and 5 in the beta-chains. The relation between Pinnipedia and Arctoidea is discussed.

Blood viscosity in phocid seals: possible adaptations to diving.

1. Mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) of phocid seal red blood cells (RBC) are elevated compared to those of most terrestrial mammalian species. The influence of these characteristics on blood flow was revealed by viscosity (VIS) measurements. 2. RBC morphology and VIS of whole blood from 7 harbor seals and 5 northern elephant seals were compared with blood of the domestic pig. Samples were analysed for RBC count, white blood cell (WBC) count, total plasma proteins, hematocrit (HCT), MCV and MCHC. Viscosity measurements were made at shear rates from 11.5 to 230.4 s-1 on a Wells-Brookfield cone-plate viscometer at 37 degrees C. 3. Mean values for HCT (%), MCV (micron 3) and MCHC (%) were, respectively: elephant seal: 57, 176, 44; harbour seal: 53, 105, 38; domestic pig: 28, 54, 34. Pig blood was reconstituted to match seal blood HCTs. VIS determinations showed that seal and pig blood conform to the general mammalian dependence of VIS upon shear rate and HCT. 4. Seal blood VIS was 28% (harbour seal) and 16% (elephant seal) less than pig blood VIS at low shear (P less than 0.05). Seal blood carried more hemoglobin per unit volume than did pig blood reconstituted to the same HCT. Fewer, larger RBC with higher MCHC, and hence elevated oxygen storage, accompanied by reduced VIS and reduced flow resistance near stasis suggests that this feature of phocid seal blood is an adaptation to circulatory redistribution during long dives.

Electrophoretic transferrin variation in fur seals (Arctocephalus spp.) at Marion Island.

1. Variation in transferrin types were investigated in sympatric populations of two fur seal species which are undergoing limited hybridization at Marion Island. 2. Vertical polyacrylamide gel electrophoresis of serum was performed to demonstrate the transferrin types. 3. The two species appear fixed for alternative alleles: A. tropicalis being homozygous for the fast allele, and A. gazella (with one exception) being homozygous for the slow allele, indicating low gene flow between these two species. The single hybrid tested was homozygous for the slow allele. 4. The use of electrophoretically determined transferrin variation holds promise for the investigation of these and other sympatric fur seal populations.

The phospholipid and fatty acid compositions of seal platelets: a comparison with human platelets.

1. Platelet phospholipid compositions were studied in four species of phocid seals consuming herring or herring and shrimp and in human subjects consuming a normal mixed diet. 2. There were no major differences in platelet phospholipid, cholesterol and protein levels between different species of seal nor between seals and human subjects, nor in the relative abundance of the individual types of phospholipid. 3. The seal platelet phospholipids (phosphatidylcholine (PC) and phosphatidylethanolamine (PE), were greatly enriched in the omega 3 fatty acid, eicosapentaenoic acid (EPA) and depressed in arachidonic acid (AA) relative to the corresponding human platelet phospholipids. 4. Much less accumulation of EPA in phosphatidylserine (PS) and phosphatidylinositol (PI) was found. 5. The EPA contents of the individual seal platelet phospholipids exhibited considerable differences (including EPA discrimination from PI) but gave patterns which were generally similar to those reported for human volunteers consuming fish/fish oils enriched in EPA. 6. These results suggest that the seal platelet may be a useful model for studying the metabolism and function of the omega 3 fatty acids, such as EPA, in relation to platelet reactivity, phospholipid turnover and the formation of AA- and EPA-derived eicosanoids.

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.

Fatty acid metabolism in fasting elephant seal pups.

The turnover of two plasma free fatty acids (FFA) were measured in 5 northern elephant seal pups (Mirounga angustirostris) after approximately 2 months of post-weaning fasting. Turnover was determined using simultaneous bolus injections of 14C-palmitate, 3H-oleate and Evans blue (EB) administered via an indwelling extradural intravertebral catheter. At this time in their natural fast, the seals exhibited plasma FFA levels and turnover values higher than reported for other marine mammals and most terrestrial carnivores. There was no consistent difference between plasma FFA turnover measured by palmitate or oleate tracers. The results imply that FFA metabolism is the primary source of energy during fasting. This is interesting in light of previous observations of minimal ketoacid accumulation and low levels of glucose and protein energy production during fasting in these juvenile seals.

Blood levels of thyroid hormones and certain metabolites in relation to moult in the harp seal (Phoca groenlandica).

1. Changes in blood levels of thyroxine (T4), triiodothyronine (T3), free fatty acids (FFA), glucose, pyruvic acid and lactic acid in the harp seal during moult, were studied. 2. Serum levels of both T4 and T3 showed significant increase in the moult phase from that in the pre-moult phase. While T4 level continued to remain high during the early and late post-moult phases, T3 level dropped in the latter two phases to the same low pre-moult level. 3. The T3/T4 ratio was significantly higher during the pre-moult phase than that in all the other phases. It is suggested that the high pre-moult T3/T4 ratio marks the initiation of moult. 4. There were no significant changes in the levels of the metabolites studied except that of FFA which was highest in the moult phase indicating the hormonal basis of lipid mobilization.

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.

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.