Evaluation of Aqueous Humor as a Surrogate for Serum Biochemistry in California Sea Lions (Zalophus californianus)
Abstract
Antemortem changes in serum chemistries are frequently required to support a postmortem diagnosis, although these data are often unavailable. In humans and domestic animals, analysis of aqueous humor collected postmortem has been used to estimate antemortem serum chemistry values, toxin levels and time of death.2,3,5,6 The aim of this study was to evaluate the use of postmortem aqueous humor in estimating serum chemistry in California sea lions (Zalophus californianus).
The objective was to compare biochemical parameters in aqueous humor at three time points after death to antemortem serum biochemical values to test the hypothesis that aqueous humor could be used to diagnose renal failure in California sea lions. Serum and aqueous humor samples were collected from 30 sea lions euthanized due to poor prognosis.4 Blood was collected from the subclavian vein within one minute of death using an 18G 1.5in needle directly into a serum separator tube. Aqueous humor was obtained from the left eye within five minutes of death by a trans-scleral approach at the limbus using a sterile 6ml syringe and a 20G 1in needle. Each animal was examined postmortem and the head was preserved at ambient temperature. Aqueous humor was collected from the right eye either 24 or 48 hours after death. All samples were stored at 4°C and analyzed within 24 hours of collection using an automated chemistry analyzer by bichromatic photometry and potentiometry (Alfa Wasserman, NJ). Parameters measured are listed in Tables 1 and 2. Serum and aqueous humor collected from nine animals diagnosed with renal failure were evaluated for antibodies to Leptospira using the Microscopic Agglutination Test.1 Linear least squares regression analysis was used to test for differences among biochemical parameters in serum and aqueous humor collected at time of death, 24 and 48 hours after death.
Mean parameter levels from serum and aqueous humor are presented in Tables 1 and 2. For blood urea nitrogen, creatinine, sodium, chloride and magnesium there was a significant positive linear relationship between serum and T0 and T24 aqueous humor levels (p<0.05 and r2 >0.80 in all cases). For aqueous humor sampled after 48 hours, the relationship was only significant for blood urea nitrogen and creatinine. For the remainder of the parameters the relationship between serum and aqueous humor chemistries was not significant. Potassium levels increased in aqueous humor collected 24 hours (paired samples t-test, n=20, p<0.001) and 48 hours (paired samples t-test, n=8, p<0.001) after death. All tested sera were positive for L. interrogans serovar pomona with titers greater than 1:25,600. Titers in aqueous humor were 0 (n=4), 1/200 to 1/400 (n=3) and 1/6400 to 1/12800 (n=2). No hemoglobin was found in any of the aqueous samples, ruling out presence of blood in the aqueous secondary to sample collection as a cause for these titers.
These results indicate that an accurate estimate of serum blood urea nitrogen and creatinine can be made from the analysis of aqueous humor at necropsy of California sea lions within 48 hours of death, facilitating the diagnosis of renal failure, (one of the commonest cause of death in wild sea lions), in beached carcasses.
Table 1. Mean ± SD concentrations and activities of biochemical parameters in serum and aqueous humor of California sea lions at time of death (T0) and 24 hours after death (T24).
Values in bold have a significant positive linear relationship at p>0.05.
|
Biochemical
parameter |
Concentration in serum
at T0 (n=20) |
Concentration in aqueous
at T0 (n=20) |
Concentration in aqueous
at T24 (n=20) |
|
Total iron (μg/dL) |
112 ± 42 |
70 ± 53 |
24 ± 28 |
|
Cholesterol (mg/dL) |
237 ± 103 |
6 ± 9 |
8 ± 13 |
|
Triglycerides (mg/dL) |
268± 311 |
11 ± 6 |
33 ± 13 |
|
γGlutamyl tranferase (U/L) |
228 ± 143 |
10 ± 24 |
10 ± 19 |
|
Alanine transaminase (U/L) |
67 ± 37 |
17 ± 5 |
11 ± 5 |
|
Aspartate transaminase (U/L) |
79 ± 132 |
23 ± 18 |
21 ± 31 |
|
Alkaline phosphatase (U/L) |
57 ± 41 |
3 ± 3 |
4 ± 3 |
|
Total bilirubin (mg/dL) |
0.8 ± 0.8 |
0.9 ± 0.6 |
0.6 ± 0.4 |
|
Glucose (mg/dL) |
125 ± 101 |
126 ± 70 |
12 ± 30 |
|
Phosphorus (mg/dL) |
13 ± 6 |
7 ± 4.9 |
9 ± 4.2 |
|
Total protein (g/dL) |
9 ±1 |
0.7 ± 0.7 |
0.5 ± 0.9 |
|
Blood urea nitrogen (mg/dL) |
212 ± 175 |
224 ± 193 |
228 ± 193 |
|
Creatinine (mg/dL) |
7 ± 8 |
5.1 ± 6 |
5 ± 5.8 |
|
Ca (mg/dL) |
8 ± 1 |
6.2 ± 0.7 |
7 ± 0.7 |
|
Na (mmol/L) |
158± 14 |
149 ± 15 |
147 ± 14 |
|
K (mmol/L) |
5 ± 1.5.1 |
6.3 ± 1.8 |
12.2 ± 2.3 |
|
Cl (mmol/L) |
116.7 ± 13.1 |
118 ± 17 |
121 ± 15 |
|
Mg (mg/dL) |
3.6 ± 1.7 |
2 ± 1.3 |
2.5 ± 1.1 |
|
Albumin (g/dL) |
2.8 ± 0.5 |
0.3 ± 0.3 |
0.3 ± 0.4 |
|
Creatine kinase (U/L) |
1429 ± 2916 |
30 ± 58 |
273 ± 727 |
Table 2. Mean ± SD concentrations and activities of biochemical parameters in serum and aqueous humor of California sea lions at time of death (T0) and 48 hours after death (T48).
Values in bold have a significant positive linear relationship at p>0.05.
|
Biochemical
parameter |
Concentration in serum
at T0 (n=10) |
Concentration in aqueous
at T0 (n=10) |
Concentration in aqueous
at T48 (n=10) |
|
Total iron (μg/dL) |
124 ± 45 |
18 ± 43 |
12 ± 15 |
|
Cholesterol (mg/dL) |
221 ± 79 |
2 ± 0.5 |
4 ± 2 |
|
Triglycerides (mg/dL) |
119 ± 164 |
10 ± 4 |
59 ± 24 |
|
γGlutamyl tranferase (U/L) |
285 ± 290 |
2 ± 1 |
3 ± 1 |
|
Alanine transaminase (U/L) |
70 ± 55 |
17 ± 7 |
12 ± 5 |
|
Aspartate transaminase (U/L) |
80 ± 114 |
17 ± 17 |
16 ± 11 |
|
Alkaline phosphatase (U/L) |
50 ± 25 |
1 ± 1 |
2 ± 2 |
|
Total bilirubin (mg/dL) |
0.9 ± 0.8 |
0.5 ± 0.5 |
0.7 ± 0.5 |
|
Glucose (mg/dL) |
135 ± 29 |
115 ± 54 |
19 ± 25 |
|
Phosphorus (mg/dL) |
12 ± 7 |
4 ± 4 |
10 ± 4 |
|
Total protein (g/dL) |
8.2 ± 1.5 |
0.2 ± 0.3 |
0.3 ± 0.2 |
|
Blood urea nitrogen (mg/dL) |
173 ± 142 |
164 ± 155 |
173 ± 163 |
|
Creatinine (mg/dL) |
4.7 ± 5.4 |
3 ± 3.3 |
3.9 ± 3.7 |
|
Ca (mg/dL) |
8.2 ± 0.8 |
6.6 ± 0.8 |
7.2 ± 0.5 |
|
Na (mmol/L) |
167 ± 18 |
157 ± 15 |
151 ± 14 |
|
K (mmol/L) |
5 ± 1.6 |
5.4 ± 1.5 |
14.7 ± 2.7 |
|
Cl (mmol/L) |
128 ± 26 |
126 ± 17 |
127 ± 14 |
|
Mg (mg/dL) |
3.6 ± 1.5 |
1.4 ± 0.9 |
1.8 ± 0.9 |
|
Albumin (g/dL) |
2.8 ± 0.6 |
0.6 ± 1.6 |
0.5 ± 0.9 |
|
Creatine kinase (U/L) |
613 ± 514 |
5 ± 6 |
221 ± 260 |
Acknowledgements
We thank the veterinary technicians and husbandry staff (D. Wickham, A. Makie, M. Blascow) of The Marine Mammal Center for their constant assistance. The care of stranded marine mammals and the opportunity of research would not be possible without the many dedicated volunteers at The Marine Mammal Center.
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