Aquatic Mammals 2013, 39(4), 330-334, DOI 10.1578/AM.39.4.2013.330
Effects of Freeze-Thaw Cycle on Urine Values from
Bottlenose Dolphins (Tursiops truncatus)
Risa L. Daniels, Cynthia R. Smith, and Stephanie Venn-Watson
The National Marine Mammal Foundation, San Diego, CA 92016, USA
E-mail: [email protected]
Abstract
Urinalysis is a valuable tool for assessing the
renal health of marine mammals. While retrospective and population renal health studies often use
frozen urine samples, it has not been determined
if the freeze-thaw process alters urine values from
bottlenose dolphins (Tursiops truncatus). The primary objective of our study was to compare the
values of 38 fresh and frozen paired urine samples
collected from 20 bottlenose dolphins at the U.S.
Navy Marine Mammal Program. Paired t-tests
and chi-squared tests were conducted to assess
the effects of storage at -80° C for 2 to 301 d,
and a subsequent thaw on urine specific gravity;
pH; creatinine; protein:creatinine ratio; quantitative protein; uric acid; uric acid:creatinine ratio;
and categorical characterizations of color, clarity,
glucose, ketones, occult blood, protein levels, and
crystals. The freeze-thaw cycle decreased urinary
pH and increased urinary uric acid (p = 0.04 and
0.02, respectively). There were no other significant changes in urine variable values, including
urinary uric acid concentration by grams of creatinine, when comparing fresh and frozen-thawed
urine samples. Urinary uric acid concentration
by grams of creatinine is the most accurate uric
acid measurement when frozen-thawed samples
are used. Urinary pH should be measured in fresh
samples to avoid falsely decreased pH.
Key Words: cetacean, urine storage effects,
bottlenose dolphin, Tursiops truncatus, urinalysis,
temperature
Introduction
Bottlenose dolphins (Tursiops truncatus) are susceptible to urate nephrolithiasis, and urinalyses
are being used to identify risk factors for nephrolith formation (Venn-Watson et al., 2010a, 2010b).
Retrospective and population studies may require
that urinalyses be performed on frozen samples
that have been stored for various periods of time.
Previous studies of terrestrial mammalian urine
have demonstrated that storage time and temperature may or may not affect urinary proteins,
pH, and uric acid; and rarely affect urine creatinine and specific gravity (Miki & Sudo, 1998;
Klasen et al., 1999; Schultz et al., 2001; Albasan
et al., 2003). Further, crystal formation that occurs
during storage of urine from terrestrial mammals
with uroliths can change uric acid values (Bartges
et al., 1996; Wells et al., 2004). In our study, we
used urine samples collected from bottlenose dolphins at the U.S. Navy Marine Mammal Program
(MMP) to test the null hypothesis that there would
be no significant differences in urine values when
comparing samples that were submitted for analysis on the day of collection and samples that were
submitted after being frozen at -80° C and thawed.
Methods
Sample Collection, Storage, and Preparation
Thirty-eight urine samples were collected opportunistically from 20 bottlenose dolphins at the MMP
in 120-ml sterile plastic specimen cups using a
variety of methods, including catheterization,
manual bladder expression, and free catch. Both
manually expressed and free catch samples were
collected mid-stream. Gross examination of urine
for color and clarity was performed immediately,
and covered samples were refrigerated within
30 min. Refrigerated samples were re-aliquoted
into two sterile 15-ml conical tubes. The first tube
was refrigerated and shipped cold via same-day
courier to a local reference laboratory. Time from
collection to processing was less than 12 h for all
fresh samples. The remaining sample was immediately frozen at -80° C for between 2 and 301 d. As
samples were collected opportunistically, sample
size for various specific time ranges was not adequate for analyses. Frozen samples were thawed
on ice and shipped the same day cold, via courier,
to the reference laboratory for analysis.
Urinalyses
At the reference laboratory, the Clinitek Atlas 10
reagent pak for urine chemistry (Bayer Corporation,
Elkhart, IN, USA [Bayer was acquired by Siemens
in January 2007.]) was used for each sample to
determine pH, color, clarity, and presence of the
following analytes: glucose, ketones, occult blood,
protein, bacteria, and crystals. Semi-automated
reflectance reading of dipsticks was performed
using the Clinitek Atlas Urine Chemistry Analyzer
(Bayer Corporation). Urine specific gravity was
determined by refractive index both manually with
a refractometer and by the Clinitek Atlas. A sulfosalicylic acid precipitation test (SSA) was used
for validation of any proteinuria detected by the
Clinitek Atlas. The Olympus AU5400 (Mishima
Olympus Company, Ltd, Olympus Corporation,
Tokyo, Japan [Olympus was acquired by BeckmanCoulter in August 2009.]) was used to analyze creatinine, protein, and uric acid colorimetrically. A
modification of the Jaffe procedure was utilized
with OSR6178 and OSR6678 reagents (Mishima
Olympus Company, Ltd) to determine creatinine levels (Jaffe, 1886). A colorimetric method
using Pyrogallol red and molybdate was utilized
with OSR6170 and OSR6270 reagents, also by
Olympus, for protein analysis. The linear range for
this method is to 15 mg/dL. The uric acid reagent
OSR6698 was utilized in a modification of the
Fossati method to determine uric acid level in the
urine (Fossati et al., 1980). Protein:creatinine and
uric acid:creatinine ratios were calculated.
Data Analysis
Data were analyzed using SAS software (Release 8e,
SAS Institute, Inc., Cary, NC, USA). Significance
was defined as a p value ≤ 0.05 for all tests.
Descriptive analyses were conducted to describe the
study population, including the number of animals
by species, number of samples, method of urine
collection, urine collection dates, sample submission dates, and time between sample collection and
submission (i.e., storage time). Paired t-tests were
used to compare the mean values of the following
urine variables by samples that were fresh or frozen-thawed: specific gravity, pH, protein:creatinine
ratio, quantitative protein, creatinine, uric acid, and
uric acid:creatinine ratio. Chi-squared analyses
were run on the following urine variables to compare the frequencies of categorical values by fresh
or frozen samples: color, clarity, glucose, ketones,
occult blood, protein, and crystals.
Results
A total of 38 fresh-frozen urine sample pairs were
collected and submitted between December 2007
and June 2009 from 20 bottlenose dolphins. Of
samples with known methods of collection, urine
was collected by catheterization (16, 43.2%),
manual bladder expression (3, 8.1%), and free
catch (18, 48.7%). The mean and median time differences of sample submissions between fresh and
frozen-thawed samples were 77 d (SD = 72 d) and
64 d (range 2 to 301 d), respectively. Mean and
frequency comparisons of urine values between
fresh and frozen samples are provided in Tables 1
& 2. Frozen-thawed samples had lower pH with a
mean change of -0.05 (range -0.5 to 0) and higher
urinary uric acid (mg/dl), with a mean change of
+2.5 (range -6.2 to 23). A total of 73% of fresh
samples had no detectable crystal compared to
58% of frozen-thawed samples (p = 0.05).
Discussion
Marine mammal urine samples are used to assess
renal health, characterize osmoregulation, determine reproductive cycles, assess potential effects
of ecosystem contaminants, and detect marine
Table 1. Comparisons of urine values between fresh and frozen-thawed samples collected from bottlenose dolphins (Tursiops
truncatus) (n = 38); median storage time was 64 d (range 2 to 301 d) at -80º C
Mean (SD)
Median (range)
Mean (SD)
Median (range)
Paired
t-test
p value
1.031 (0.008)
6.3 (0.3)
269 (127)
1.030 (1.020-1.050)
6.5 (6.0-7.0)
253 (64-611)
1.032 (0.008)
6.3 (0.3)
265 (135)
1.030 (1.020-1.050)
6.5 (6.0-7.0)
253 (85-629)
0.25
0.04
0.52
35 (20)
143 (77)
22 (18)
228 (252)
31 (8-92)
126 (33-412)
16 (1-73)
126 (6-964)
39 (24)
148 (81)
24 (20)
235 (263)
30 (9-100)
134 (38-427)
16 (0.1-79)
101 (0.6-974)
0.15
0.25
0.02
0.26
Fresh urine
Urine variable
Specific gravity
pH
Protein:creatinine (mg/g
creatinine)
Quantitative protein (mg/dl)
Creatinine (mg/dl)
Uric acid (mg/dl)
Uric acid:creatinine (mg/g
creatinine)
Frozen-thawed urine
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Daniels et al.
Table 2. Comparisons of urine values between fresh and frozen-thawed (-80° C) samples collected from 20 bottlenose
dolphins (38 sample pairs); median storage time was 64 d (range 2 to 301 d).
Urine variable
% fresh sample
n = 38
% frozen sample
n = 38
Color
Yellow
Dark yellow
79
21
87
13
Clarity
Clear
Cloudy
Turbid
76
16
8
74
18
8
100
100
100
100
100
100
Glucose (negative)
Ketones (negative)
Nitrite (negative)
p value
0.36
0.85
Occult blood
Negative
Trace
Small
Moderate
Large
68
8
5
8
11
71
5
11
0.0
13
Protein
Negative
Trace
30 mg/dl
100 mg/dl
55
26
16
3
43
32
22
3
Crystal type
None
Amorphousb
Calcium oxalate
Triple phosphate
Other
66
11
13
3
8
58
13
16
0
13
Crystal quantity
None
Few (1-5)
Moderate (6-15)
Many (> 15)
73
24
3
0
58
37
0
5
NAa
NA
NA
0.90
0.99
0.55
0.05
Not applicable
Urate or phosphate
a
b
toxins (Ortiz, 2001; Gulland et al., 2002; Roebeck
et al., 2005; Venn-Watson et al., 2010a, 2010b).
This study focused on the use of comprehensive
urinalyses to study individual animal and population renal health. While individual case workups
typically involve fresh urine samples, population
health assessments and retrospective studies may
require frozen samples. As such, it is important to
determine the potential effects of the freeze-thaw
cycle on urinalysis values. In our study, we did not
find any significant changes in urine creatinine,
specific gravity, proteins, or uric acid concentration (mg/g creatinine) when comparing fresh and
frozen-thawed bottlenose dolphin urine samples
stored at -80° C. We did find lower pH and higher
uric acid (mg/dl) in urine samples that were frozen-thawed compared to fresh samples.
Previous studies have assessed the effects of
storage temperature and time on urine creatinine, specific gravity, protein, and pH of terrestrial mammals. Urinary creatinine appears to be
stable in humans and demonstrates no significant
change when stored at 4° C for 1 wk, -4° C for
2 mo, or -20° and -70° C for 6 to 8 mo (Spierto
et al., 1997; Miki & Sudo, 1998). Spierto et al.
(1997) concluded “that in all but extreme cases
urine creatinine is virtually unaffected by storage
temperature and time.” At room and refrigeration
temperatures, urine specific gravity of dogs and
cats was not affected after 24 h storage (Albasan
et al., 2003). Many studies have been conducted
to assess the ability to accurately detect microalbuminuria in frozen urine of diabetic patients
(Klasen et al., 1999; Schultz et al., 2001). While
urinary proteins did not appear stable at -20° C,
urine protein values have remained valid at 4° C
for up to 4 wks and longer at -70° C (Klasen et al.,
1999; Schultz et al., 2001). These studies support
our findings that bottlenose dolphin urine samples
stored at low temperatures (-80° C) will maintain
stable creatinine levels, specific gravity, and protein levels.
Unlike creatinine and specific gravity, urinary
pH in terrestrial species may increase falsely due
to degradation of nitrogenous urine analytes when
stored at room or refrigerator temperatures; urinary pH in humans appeared to be stable, however,
at -20° C (Cook et al., 2007). In our study, urinary
pH decreased slightly in samples that were frozenthawed. Bottlenose dolphins eat high purine diets.
As such, higher levels of protein-based metabolites in the urine, including ammonium and uric
acid, compared to humans are expected. Given
the concurrent increase in measurable urinary uric
acid in frozen-thawed samples from our study, it is
likely that increased uric acid was associated with
a more acidic pH reading.
Reports related to the effects of storage temperature and time on uric acid in terrestrial mammals are less straightforward than those focusing
on creatinine, pH, and specific gravity. One study
reported that undiluted human urine samples
may have falsely decreased uric acid levels over
time at 4° and 20° C if urine has both high uric
acid concentrations and low pH (Yamakita et al.,
2000). Another study involved urine samples from
dogs with ammonium urate uroliths. Pure breeds
required a 1:20 dilution to maintain valid urine
uric acid results if stored for 1 to 12 wks at -20° C.
Interestingly, samples from mixed breed dogs
without uroliths did not require dilution to maintain valid urine uric acid levels (Bartges et al.,
1996). Our study demonstrated an increase in urinary uric acid, though not after correcting for creatinine, when comparing fresh and frozen-thawed
samples. Interestingly, our study population has a
high prevalence of ammonium acid urate nephrolithiasis (Venn-Watson et al., 2010a). Follow-up
research is needed to assess the need for diluting
frozen urine samples for accurate uric acid levels.
333
Though interassay variation may be considered
a limitation, the MMP has established normal reference ranges for 27 urine analytes in bottlenose
dolphin urine. These baselines are the result of
over 500 MMP bottlenose dolphin urine samples,
analyzed at the reference laboratory, giving us
high confidence in reliability and comparability
of results. Preliminary analyses tested associations
between storage time and measured differences
of urine values between fresh and frozen-thawed
(-70° C) samples collected from MMP bottlenose
dolphins (24 sample pairs) and California sea lions
(6 sample pairs). Median storage time was 84 d.
Specific gravity, pH, protein:creatinine ratio,
quantitative protein, creatinine, uric acid, and uric
acid:creatinine ratio were analyzed. The amount of
time that samples were in storage did not significantly affect differences between fresh and frozenthawed samples. Future analyses, including comparison of results based on various freezing time
periods, will only strengthen confidence in utilizing frozen-thawed bottlenose dolphin urine.
In conclusion, bottlenose dolphin urinary creatinine, specific gravity, proteins, and uric acid
concentration by grams of creatinine appear to be
stable when samples are stored at -80° C for up to
301 d and thawed. Caution needs to be used if urinary pH and uric acid (mg/dl) without creatinine
are measured in frozen-thawed samples.
Acknowledgments
The authors thank the animal care staff of the U.S.
Navy Marine Mammal Program and the Army
animal care specialists for sample collection and
processing.
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