Serum symmetric dimethylarginine in older dogs: Reference interval and comparison of a gold standard method with the ELISA.

BACKGROUND: Serum symmetric dimethylarginine (SDMA) is used to screen for renal dysfunction in dogs. The gold standard technique for measuring SDMA, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is not widely available. Age-specific reference intervals for SDMA in older dogs are lacking. OBJECTIVES: Prospective study in older dogs to validate a commercially available LC-MS/MS method for SDMA, compare SDMA concentrations with concentrations measured using ELISA and obtain a reference interval (RI) for older dogs using both methods. ANIMALS: Client-owned older dogs undergoing health screening. METHODS: The LC-MS/MS method was analytically validated (limit of detection, precision, and linearity). Serum was sent cooled overnight for ELISA or was frozen at -80°C until batch analysis using LC-MS/MS. Results of LC-MS/MS and ELISA were compared and RIs for older dogs were calculated according to international guidelines. RESULTS: The LC-MS/MS method showed good linearity (r2 = .99) and precision (coefficient of variation <10%), with a laboratory RI between 8.0 and 14.0 µg/dL. Paired measurements were available from 118 different dogs. Median SDMA concentration were 9.4 (range, 5.0-21.2) using LC-MS/MS and 12.0 (range, 5.0-22.0) µg/dL using ELISA. Both methods significantly differed with a mean difference of 2.2 µg/dL. The RI for older dogs for LC-MS/MS was 4.4-15.0 µg/dL, and for ELISA was 6.4-17.4 µg/dL. CONCLUSIONS AND CLINICAL IMPORTANCE: The ELISA provided significantly higher SDMA concentrations compared to the validated LC-MS/MS method, indicating the need for device- or assay-specific RI. The obtained age-specific RI for SDMA is considerably higher in older dogs compared to the general laboratory RI.

Plasma and urinary metanephrine and normetanephrine concentrations using liquid chromatography with tandem mass spectrometry in healthy cats and in a cat with pheochromocytoma.

BACKGROUND: Pheochromocytoma (PCC) is rare in cats and plasma (PL) and urinary (U) metanephrines (metanephrine [MN]; normetanephrine [NMN]) measurement is rarely described in cats. OBJECTIVES: We evaluated the utility of PL and U MNs measurement in 10 healthy cats and a cat with a confirmed diagnosis of pheochromocytoma (PheoCat), using liquid chromatography with tandem mass spectrometry (LC-MS-MS). METHODS: Urine and EDTA PL samples collected from each of the 10 cats and the PheoCat were promptly stored at -80°C and remained frozen until analysis. To evaluate U MNs stability, an additional urine sample collected from the healthy cats was refrigerated for 24 hours before freezing. Urinary creatinine concentration (Creat) was assessed using the same spot urine samples to calculate U MNs-to-creatinine ratios. RESULTS: The PL-MN and PL-NMN median concentrations of the healthy cats were 2.73 and 7.02 nmol/L, respectively. The median U-MN/Creat and U-NMN/Creat ratios were 70 and 139 µg/g, respectively. The PheoCat had a PL-MN of 3.68 nmol/L, PL-NMN of 66.27 nmol/L, U-MN/Creat of 179 µg/g, and U-NMN/Creat of 1262 µg/g. The PheoCat had markedly increased concentrations of both PL and U MNs when compared to the healthy cats. No significant difference was found between U MNs measured in urine samples that underwent 24 hours of refrigeration in comparison to those that were frozen immediately. CONCLUSIONS: We report preliminary reference intervals for PL and U MNs in cats using LC-MS-MS and the potential clinical applicability of these biomarkers for the diagnosis of PCC in cats.

Organohalogenated contaminants in plasma and eggs of rockhopper penguins: Does vitellogenin affect maternal transfer?

Although many studies have investigated organohalogenated contaminants (OHCs) in yolk, little is known about the mechanisms and timing of transfer of OHCs from the female to the egg. Vitellogenin, a yolk precursor, has been suggested to play a role in this transport. We here report for the first time the temporal changes in OHC and an index of vitellogenin concentrations in female plasma from the pre-laying period to clutch completion in free-living birds: the southern rockhopper penguin (Eudyptes chrysocome chrysocome) breeding in the Falkland/Malvinas Islands. In addition, OHC concentrations in the corresponding clutches were analysed. OHC concentrations in female plasma and in the yolk of both the first (A-) and the second (B-)eggs followed a similar pattern, with hexachlorobenzene (HCB) > Σpolychlorinated biphenyls (PCBs) > Σdichlorodiphenyltrichloroethanes (DDTs) > Σmethoxylated polybrominated diphenyl ethers (MeO-PBDEs) > Σchlordanes (CHLs) > Σpolybrominated diphenyl ethers (PBDEs) ≈ Σhexachlorocyclohexanes (HCHs). The higher concentrations of MeO-PBDEs compared to PBDEs indicate a diet containing naturally-produced MeO-PBDEs. All OHC compounds except for PBDEs increased from the pre-laying period to A-egg laying and subsequently declined from A-egg laying to B-egg laying, and female plasma vitellogenin showed the same pattern. For ΣPCBs and ΣMeO-PBDEs, we found positive correlations between female plasma during A-egg laying and both eggs, and for HCB between female plasma and A-eggs only. During pre-laying, only ΣMeO-PBDEs correlated between both eggs and female plasma, and no correlations between OHC concentrations in eggs and female plasma were found during B-egg laying, highlighting that maternal transfer of OHCs is time- and compound-specific. Finally, female vitellogenin concentrations did not significantly correlate with any OHC compounds in either female plasma or eggs, and our results therefore did not confirm the suggested role of vitellogenin in the maternal transfer of OHC molecules into their eggs.

Can starling eggs be useful as a biomonitoring tool to study organohalogenated contaminants on a worldwide scale?

Large-scale international monitoring studies are important to assess emission patterns and environmental distributions of organohalogenated contaminants (OHCs) on a worldwide scale. In this study, the presence of OHCs was investigated on three continents (Europe, North America and Australasia), using eggs of starlings (Sturnus vulgaris and Sturnus unicolor) to assess their suitability for large-scale monitoring studies. To the best of our knowledge, this is the first study using bird eggs of the same species as a biomonitor for OHCs on an intercontinental scale. We found significant differences in OHC concentrations of the eggs among sampling locations, except for hexachlorocyclohexanes (HCHs). Mean concentrations of sum polychlorinated biphenyls (PCBs) in eggs ranged from 78±26 ng/glipid weight (lw) in Australia to 2900±1300 ng/g lw in the United States. The PCB profile was dominated by CB 153 and CB 138 in all locations, except for New Zealand, where the contribution of CB 95, CB 101 and CB 149 was also high. The highest mean sum polybrominated diphenyl ether (PBDE) concentrations were found in Canada (4400±830 ng/g lw), while the lowest mean PBDE concentrations were measured in Spain (3.7±0.1 ng/g lw). The PBDE profile in starling eggs was dominated by BDE 47 and BDE 99 in all countries, but in Belgium, the higher brominated PBDEs had a higher contribution compared to other countries. For the organochlorine pesticides (OCPs), dichlorodiphenyltrichloroethanes (DDTs) ranged from 110±16 ng/g lw in France to 17,000±3400 ng/g lw in New Zealand, while HCHs and hexachlorobenzene were generally in low concentrations in all sampling locations. Chlordanes were remarkably high in eggs from the United States (2500±1300 ng/g lw). The OCP profile in all countries was largely dominated by p,p'-DDE. In general, the worldwide trends we observed in starling eggs were in accordance with the literature on human and environmental OHC data, which suggests that there is potential for using starling eggs as a biomonitoring tool on a large geographical scale.

Organohalogenated contaminants in eggs of rockhopper penguins (Eudyptes chrysocome) and imperial shags (Phalacrocorax atriceps) from the Falkland Islands.

In this study, we evaluated the use of seabird eggs of two common bird species from the Falkland Islands as bioindicators of contamination with organohalogenated contaminants (OHCs). We compared contamination levels and profiles of different OHCs between eggs of the rockhopper penguin (Eudyptes chrysocome) and the imperial shag (Phalacrocorax atriceps). In addition, laying order effects on OHC concentrations and profiles were also investigated in both species. For polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) as well as polybrominated diphenyl ethers (PBDEs), concentrations were significantly lower in eggs of rockhopper penguins (27.6±0.70 ng/g lw, 56.5±1.33 ng/g lw and 0.98±0.04 ng/g lw, respectively) compared to the imperial shags (140±5.54 ng/g lw, 316±11.53 ng/g lw, 1.92±0.15 ng/g lw, respectively). On the other hand, 2'MeO-BDE 68 and 6MeO-BDE 47, two brominated compounds of reported natural origin, were significantly higher in the penguin eggs (0.55±0.05 ng/g lw and 7.01±0.64 ng/g lw, respectively) compared to the shag eggs (0.17±0.03 ng/g lw and 0.50±0.06 ng/g lw, respectively). In addition, PCB, OCP and PBDE contamination profiles differed markedly between the two species. Various factors, such as diet, feeding behaviour, migratory behaviour and species-specific metabolism, may be responsible for the observed results. For both rockhopper penguins and imperial shags, PCB, OCP and PBDE concentrations and profiles did not significantly change in relation to the laying order. This suggests that, for both species, any egg of a clutch is useful as a biomonitoring tool for OHCs. Although our results showed that OHCs have also reached the Falkland Islands, concentrations were relatively low compared to other studies. However, future monitoring may be warranted to assess temporal trends of different OHCs.

Endocrine disrupting, haematological and biochemical effects of polybrominated diphenyl ethers in a terrestrial songbird, the European starling (Sturnus vulgaris).

We exposed female European starlings (Sturnus vulgaris) during a 6month period to environmentally relevant concentrations of a pentabromodiphenyl ether (Penta-BDE) mixture (about 1740µg/kg body weight) through subcutaneous implants, and examined endocrine disruptive, haematological and biochemical effects. To investigate endocrine disrupting effects of PBDEs, testosterone, estradiol and thyroid hormones (T(3) and T(4)) were measured in the plasma. We found no significant differences between the control and exposed groups for any of the hormones. However, 14days after implantation, T(3) concentrations tended to be lower in the exposed group compared to the control group. For albumin, our results suggest that short term exposure of PBDEs may result in a negative effect on albumin, while the opposite was observed on a longer term (6months after implantation). However, from the statistical analysis, effects on albumin disappeared when excluding females that laid eggs. These results demonstrate that observed effects during the breeding season should be interpreted carefully when the breeding status of females is unknown. There were no significant differences found between the control and exposed groups for the other biochemical parameters (total protein, uric acid, cholesterol, aspartate transaminase, total antioxidative capacity, high-density lipoproteins and creatine kinase) and haematological parameters (amount of red blood cells, haemoglobin concentration, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin). All together, our results suggest that European starlings, similar to other passerine species, may be less sensitive to the effects of organohalogenated pollutants, such as PBDEs, than other bird species.

The use of blue tit eggs as a biomonitoring tool for organohalogenated pollutants in the European environment.

In the present study, large scale geographical variation in the occurrence of organohalogenated pollutants (OHPs) was investigated throughout Europe using eggs of a terrestrial resident passerine species, the blue tit (Cyanistes caeruleus). Blue tit eggs from 10 sampling locations, involving suburban, rural and remote areas, in 7 European countries were collected and analysed. Sum polychlorinated biphenyl (PCB) levels ranged from 150ng/g lipid weight (lw) to 2003ng/g lw. Sum polybrominated diphenyl ethers (PBDEs) ranged from 3.95ng/g lw to 114ng/g lw. As expected, PCB and PBDE concentrations were significantly higher in the sampled suburban locations compared to the rural and remote locations. Sum organochlorine pesticides (OCPs) ranged from 122ng/g lw to 775ng/g lw. OCP concentrations were, against the expectations, found to be lower in the rural sampling locations compared to the other locations. Contamination profiles of PCBs, PBDEs and OCPs differed also among the sampling locations, which may be due to local contamination sources. Finally, we compared the results of this study with previously reported OHP concentrations in the eggs of a closely related species, the great tit (Parus major), from the same sampling locations in Europe. We found no differences in concentrations between the species. In addition, we found a significant, positive correlation between the sum PCB concentrations in blue tit eggs and great tit eggs, suggesting similar exposure pathways, mechanisms of accumulation and maternal transfer of PCBs. In conclusion, our results suggest the usefulness of eggs from passerine birds as a biomonitoring tool for OHPs on a large geographical scale.

Effects of laying order and experimentally increased egg production on organic pollutants in eggs of a terrestrial songbird species, the great tit (Parus major).

In this study, concentrations and profiles of organic pollutants were investigated in a passerine species with a large clutch size, the great tit (Parus major). In the first clutches, mean egg concentrations decreased significantly in relation to the laying order from 3025+/-416 ng/g lw to 2267+/-386 ng/g lw for sum PCBs and from 989+/-339 ng/g lw to 695+/-320 ng/g lw for sum DDTs. Sum PBDE concentrations also decreased in relation to the laying order from 68+/-10 ng/g lw to 53+/-11 ng/g lw, but not significantly. Although laying order effects were found, variation in concentrations within clutches was smaller than among clutches. To further investigate the impact of laying large numbers of eggs on levels and profiles of organic pollutants, initiation of replacement clutches was experimentally induced. Mean sum PCB and sum PBDE concentrations were significantly lower in eggs of replacement clutches compared to first clutches. In addition, first clutches had a higher contribution of the higher chlorinated and more persistent PCB congeners, CB 170, 180 and 183, and a lower contribution of CB 52, 95 and 149 compared to replacement clutches. Because of the differences in concentrations and profiles between the first and replacement clutches, the combined use of eggs from both the first and replacement clutches for monitoring purposes is not recommended. In conclusion, we suggest that, due to the larger variation among clutches compared to the variation within clutches, one randomly collected great tit egg from a first clutch is useful as a biomonitoring tool for organic pollutants. To our knowledge, this is the first study in which the impact of an experimentally increased clutch size on the levels and profiles of contaminants in eggs has been investigated.

An exposure study with polybrominated diphenyl ethers (PBDEs) in female European starlings (Sturnus vulgaris): toxicokinetics and reproductive effects.

We exposed female European starlings to a pentabromodiphenyl ether (Penta-BDE) mixture through subcutaneous implants, and examined levels and profiles of polybrominated diphenyl ethers (PBDEs) together with reproductive effects. Sum PBDE levels increased significantly in the serum of the exposed females from 218+/-43 to 23,400+/-2035 pg/ml. Sum PBDE concentrations in the eggs of the exposed group ranged from 130+/-12 to 220+/-37 ng/g wet weight (ww). The profile in serum after egg laying was very similar to that in eggs. There were no detectable levels of HO-PBDEs in both serum and eggs. Fewer females of the exposed group initiated egg laying compared to the control group, although the difference was not significant. In addition, egg weight and volume were significantly higher in the exposed group. These results suggest that, at the investigated exposure levels (150 microg sum PBDEs/bird), PBDEs may have a negative effect on reproductive performance.

Brominated flame retardants and organochlorines in the European environment using great tit eggs as a biomonitoring tool.

Large-scale studies are essential to assess the emission patterns and spatial distribution of organohalogenated pollutants (OHPs) in the environment. Bird eggs have several advantages compared to other environmental media which have previously been used to map the distribution of OHPs. In this study, large-scale geographical variation in the occurrence of OHPs, such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and organochlorine pesticides (OCPs), was investigated throughout Europe using eggs of a terrestrial residential passerine species, the great tit (Parus major). Great tit eggs from 22 sampling sites, involving urban, rural and remote areas, in 14 European countries were collected and analysed (5-8 eggs per sampling site). The environmentally most important congeners/compounds of the analysed pollutants were detectable in all sampling locations. For PCBs, PBDEs and OCPs, no clear geographical contamination pattern was found. Sum PCB levels ranged from 143 ng/g lipid weight (lw) to 3660 ng/g lw. As expected, PCB concentrations were significantly higher in the sampled urban compared to the remote locations. However, the urban locations did not show significantly higher concentrations compared to the rural locations. Sum PBDEs ranged from 4.0 ng/g lw to 136 ng/g lw. PBDEs were significantly higher in the urbanized sampling locations compared to the other locations. The significant, positive correlation between PCB and PBDE concentrations suggests similar spatial exposure and/or mechanisms of accumulation. Significantly higher levels of OCPs (sum OCPs ranging from 191 ng/g lw to 7830 ng/g lw) were detected in rural sampling locations. Contamination profiles of PCBs, PBDEs and OCPs differed also among the sampling locations, which may be due to local usage and contamination sources. The higher variance among sampling locations for the PCBs and OCPs, suggests that local contamination sources are more important for the PCBs and OCPs compared to the PBDEs. To our knowledge, this is the first study in which bird eggs were used as a monitoring tool for OHPs on such a large geographical scale.

Interspecific differences in concentrations and congener profiles of chlorinated and brominated organic pollutants in three insectivorous bird species.

We investigated the accumulation of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and organochlorine pesticides (OCPs) in eggs of three insectivorous bird species, the great tit (Parus major), the Northern lapwing (Vanellus vanellus) and the Mediterranean gull (Larus melanocephalus), near the harbour of Antwerp (Belgium). Our results show that lapwing eggs had the highest median concentrations of PCBs (4358 ng/g lw) and PBDEs (109 ng/g lw). Mediterranean gulls feed during breeding on ground-dwelling invertebrates on agricultural fields, which is reflected in higher OCP concentrations in eggs (1235 ng/g lw). Apart from differences in accumulation, also interspecific differences in contaminant profiles were investigated. Significant differences among species were found in the profile of PCBs, PBDEs and OCPs. These differences could be attributed to differences in diet, behaviour and metabolic capacity. Interestingly, the OCP profile in lapwing eggs deviated extremely from the two other species. In both great tit and Mediterranean gull eggs p,p'-DDE was by far the most important compound, whereas in lapwing eggs hexachlorobenzene, oxychlordane, trans-nonachlor and even p,p'-DDT were relatively more abundant than p,p'-DDE. The high p,p'-DDT/p,p'-DDE ratio has previously been described in lapwings, which suggests that low p,p'-DDE accumulation in eggs might be inherent for this species.

Maternal transfer of organochlorines and brominated flame retardants in blue tits (Cyanistes caeruleus).

Although eggs have frequently been used as a biomonitoring tool for contamination with organohalogenated pollutants (OHPs), few studies have investigated the processes of maternal transfer in birds. Here, maternal transfer of polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and polybrominated diphenyl ethers (PBDEs) was investigated through comparison of the concentrations and profiles between whole homogenised female blue tits (Cyanistes caeruleus) and their eggs. In addition, we examined if there was an effect of laying order on the concentrations of PCBs, OCPs and PBDEs. PCBs were the most abundant contaminants in female blue tits and their eggs, followed by OCPs and PBDEs. Among the OCPs, p,p'-DDE was the most dominant compound and accounted for more than 80% of the sum OCPs. Egg concentrations decreased significantly in relation to the laying order from 1623+/-148 ng/g lipid weight (lw) to 1040+/-47 ng/g lw for the sum PCBs, from 342+/-24 ng/g lw to 235+/-17 ng/g lw for the sum OCPs and from 49+/-5 ng/g lw to 27+/-5 ng/g lw for the sum PBDEs. When reviewing all studies investigating laying order effects of OHPs in birds, no clear patterns emerged, which may be due to differences in study species and methodology among studies. Despite the fact that there were laying order effects in blue tit clutches, the variance in concentrations of PCBs and PBDEs was larger among clutches than within clutches. Variance in OCP concentrations among clutches was similar to the variance within clutches. These results suggest that one randomly collected blue tit egg from a clutch is useful as biomonitoring tool for PCBs and PBDEs, while for OCPs it is recommended to consistently use the same egg from the laying sequence as a biomonitoring tool. Lipid-normalized concentrations of sum PCBs, sum OCPs and sum PBDEs in female blue tits after clutch completion were comparable to the concentrations in the first-laid eggs. The egg/female lipid concentration ratios for sum PCBs, sum OCPs and sum PBDEs decreased significantly from 1.0+/-0.09 to 0.7+/-0.003, 1.1+/-0.1 to 0.7+/-0.02 and 1.2+/-0.14 to 0.8+/-0.05, respectively, during the course of egg laying. The profiles of the females and eggs differed significantly for the PCBs, OCPs and PBDEs. There was a higher contribution of lower chlorinated PCB congeners (CB 28, CB 52, CB 95, CB 101 and CB 110), BDE 183, p,p'-DDT and other less persistent OCPs in the females compared to their eggs. Maternal transfer in blue tits seemed to be selective for the more bioaccumulative and persistent congeners/compounds.

Variation, levels and profiles of organochlorines and brominated flame retardants in great tit (Parus major) eggs from different types of sampling locations in Flanders (Belgium).

Small-scale geographical variation in the occurrence of polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and polybrominated diphenyl ethers (PBDEs) was investigated using the eggs of a terrestrial residential songbird species, the great tit (Parus major). In addition, we investigated the influence of the type of sampling location on the presence of these pollutants. To achieve this, 10 different sampling locations in Flanders (Belgium) were classified into 3 groups based on the extent of urbanisation, industrialisation and agriculture. The higher variance among sampling locations for the levels and profiles of PCBs and OCPs, suggests that local contamination sources are more important for the PCBs and OCPs compared to the PBDEs. Levels of PCBs and PBDEs were significantly higher in the industrialised sampling locations compared to the other locations. Sum PCB and sum PBDE levels reached up to 6050 and 79 ng/g lipid weight, respectively. PCBs and PBDEs were highly positively correlated for all groups, suggesting similar exposure pathways and/or mechanisms of accumulation. Significantly higher levels of OCPs (sum OCPs up to 2683 ng/g lipid weight) were detected in the rural sampling locations situated in a residential area. This suggests that local historical usage of OCPs by inhabitants may be an important source of contamination in Flanders. Contamination profiles differed also among the sampling locations. The rural sampling locations had a higher contribution of lower brominated BDE congeners, whereas the industrialised locations had a higher contribution of higher brominated congeners. The differences in contamination profiles among the sampling locations are probably due to differences in exposure. In conclusion, our results showed that the characteristics of a sampling location influence both the levels and profiles of PCBs, OCPs and PBDEs.