The Effect of Storing Temperature and Duration on Urinary Hydration Markers

J.D. Adams1, Stavros A. Kavouras1, Evan C. Johnson 2, Lisa T. Jansen1, Catalina Capitan 3, Joseph

I. Robillard1, Andy Mauromoustakos4

1
Department of Health, Human Performance, and Recreation, University of Arkansas;
2
Department of Kinesiology and Health, University of Wyoming;

3
Universidad Hispanoamericana, San Jose, Costa Rica;

4
Department of Crop, Soil, and Environmental Sciences, University of Arkansas

Correspondent author:

Stavros Kavouras, FACSM, FACSS

Department of Health, Human Performance, and Recreation

Fayetteville, AR 72701

E-mail: kavouras@uark.edu

Phone: (479) 575-5309

Fax: (479) 575-5778

ABSTRACT

Purpose: Collection of urine samples in human studies many times involves sample storing for

later analysis. Storage temperatures and duration of storing the urine samples prior to analysis

may influence the accuracy of measured variables. The purpose of this investigation was to

quantify the effects of storage temperature, duration, and the urinary sediment on urinary

hydration markers. Methods: Thirty-six human urine samples were either analyzed fresh or

stored in 24 separate vials, six in each of the following four temperatures: 22 °C, 7 °C, -20 °C,

and -80 °C. Two of each sample stored in any given temperature, were analyzed after 1, 2, and 7

days either following vortexing or centrifugation. Each urine sample was analyzed for osmolality

(UOsm), urine specific gravity (USG), and urine color (UC). Results: At 22 °C, UOsm was

stable for 1 day but slightly increased after 2 days (+9 mmol∙kg-1, P=0.044). At 4 °C, UOsm was

stable up to 7 days (+8 mmol∙kg-1, P=0.090). At -20 °C, UOsm decreased after 1 day (-50

mmol∙kg-1, P=0.002), 2 days (-79 mmol∙kg-1, P>0.001), and 7 days (-86 mmol∙kg-1, P<0.001). At

-80 °C, UOsm decreased after 1 day (-54 mmol∙kg-1, p=0.020), 2 days (-57 mmol∙kg-1, P=0.001),

and 7 days (-59 mmol∙kg-1, P<0.001). Between agitation methods, there were significant

differences between vortex and centrifugation only in the -20 °C and -80 °C storage

environments. Conclusions:, The data indicated that urine specimens analyzed for UOsm or

USG remained stable in refrigerated environment for up to 7 days, and in room temperature for

up to 2 days. However, freezing samples significantly decreased the values of hydration markers.

Key words: urine osmolality, urine samples, hydration indexes, laboratory methods
INTRODUCTION

Urine specimens are widely used as indices of hydration (20, 21) and are being collected

and stored as part of standard protocols for large cohort studies (13, 15, 27) as well as small

experimental trials (9, 17). These specimens are often required to be processed within 3 h of

sampling or must be transported and delivered to the laboratory on ice. This protocol has been in

place in many laboratories and is based on the Clinical and Laboratory Standards Institute

(CLSI) guidelines on preservation and stability of urine (23). However, certain laboratory and

field studies may require shipping of samples for analysis at another location, as well as a delay

in the analysis for future research questions (24).

The temperature and the duration of sample storing may influence the determination of

urinary hydration markers, such as urine osmolality, urine specific gravity, and urine color (2,

19). Additionally, crystal formation that occurs at the bottom of the vial during storage of urine

may also alter urinary markers, or even metabolites such as uric acid values. However, no study

has ever examined if urine precipitation affects urine hydration markers (4, 12).

To date there have been few studies investigating the effects of time and storage

temperature on urinary hydration markers. One of the first to examine the effect of time on urine

osmolality, was a white paper from an osmometer manufacturing company, which reported good

stability up to 24 h in either room temperature (22 °C) or refrigerated temperature (4 °C, 11).

Recently, Bezuidenhout et al. (6) found urine osmolality to be extremely stable up to 36 h when

stored in a refrigerated environment (4-8 °C) with average deviations from baseline osmolality

not exceeding 2 mOsm∙kg-1 at any time point. However, the aforementioned study did not
examine the storing conditions of either a household (-20 °C) or deep freezer (-80 °C), which are

widely used when immediate analysis is not possible.

At this time there is no consensus on urine sample handling and preservation due to lack

of experimental data. Therefore, the purpose of the study was to examine the effect of storage

time and temperature, as well as the urinary sediment on urinary hydration markers.

METHODS

Thirty-six separate urine samples were collected from healthy males and females without

history of renal diseases (age: 43.7±13.3 y, height: 1.71±0.01 m, weight: 76.3±13.3 kg, body fat:

27.8±9.6 %). The study was a subset of a larger study conducted in our laboratory, and was

approved by the University’s Institutional Review Board. Within two hours of urine collection

each sample was either analyzed fresh or stored in 24 cryovials, six in each of the following four

temperatures: 22 °C, 7 °C, -20 °C, and -80 °C. Two of each sample stored in any given

temperature, were analyzed after 1 (24 h), 2 (48 h), and 7 days either following vortexing or

centrifugation. Figure 1 depicts the conditions (storing temperature, duration, and treatment) that

all urine samples were analyzed. The samples stored in 7 °C, -20 °C, and -80 °C were removed

from the stored environment to reach room temperature prior to vortexing or centrifugation for at

least 30, 60 and 90 min, respectively. It should be noted that the fresh samples were not vortexed

prior to analysis.

All 24 cryovials were analyzed for urine osmolality (UOsm), urine specific gravity

(USG), and urine color (UC). Before each sample was analyzed, the urine specimens were

vortexed for 5 seconds or centrifuged at 17,000 g for 10 minutes. UOsm was determined via
freezing point depression (Advanced Instruments 3250 osmometer; Norwood, MA) in duplicate,

or in triplicate if the osmolality readings were differed by ≥3 mmol∙kg-1. Intra-assay coefficient

of variation for the osmometer was 0.21% while inter-assay coefficient of variation was 0.38%,

respectively. USG was measured using an optical refractometer, while the samples were in room

temperature (ATAGO, Tokyo, Japan). Samples were transferred to a 15 mL glass tube for the UC

assessment by the same researcher, in a well-lit room by fluorescent light against white

background. UC was assessed by comparison to the 8-point urine color chart, with values

ranging from 1 (very light) to 8 (very dark, 3). The color was recorded as a whole number

integer.

Statistical Analyses

Regression analysis was used to show any correlation from fresh sample to stored sample

plotted with the line of identity. Due to the large number of comparisons only the three most and

least accurate estimations are graphed. Fresh sample versus stored sample values of each storage

temperature and corresponding time point were evaluated by Bland-Altman comparison method

for UOsm, USG, and UC (7). Bland-Altman analysis was employed to evaluate the agreement

between freshly analyzed sample for UOsm, USG or UC,, as the reference method, and stored

samples for each storage temperature and time point when samples were vortexed or centrifuged

prior to the analysis. Percentage of agreement was calculated as the ratio of participants who had

mean differences between the two methods within the range [M(difference) ± 1.96 SD(difference) ].

Multifactorial analysis was applied to examine differences between vortex and centrifuge

samples for each time and temperature. Statistical analysis was performed using JMP Pro

(version 12, SAS Inc., Gary, NC, USA).

RESULTS

Table 1 shows the Bland-Altman analysis for UOsm of vortexed samples. The values are

mean difference from fresh, upper and lower 95% confidence interval, and P-value. Storing the

sample in room temperature (22 °C), did not alter the UOsm compared to fresh after 1 day (mean

difference +5 mmol/kg, P=0.216), while a small but significant increase was observed after 2

days (mean difference +9 mmol∙kg-1, P=0.044) and 7 days (mean difference +19 mmol∙kg-1,

P<0.001). In the 7 °C storage, UOsm did not differ from fresh after 1 day (mean difference +3

mmol∙kg-1, P=0.467), 2 days (mean difference +4 mmol∙kg-1, P=0.349), or 7 days (mean

difference +8 mmol/kg, P=0.090). In the -20 °C environment, UOsm significantly declined after

1 day (mean difference -50 mmol∙kg-1, P=0.002), 2 days (mean difference -79 mmol∙kg-1,

P>0.001), and 7 days (mean difference -86 mmol∙kg-1, P<0.001). Lastly, in the -80 °C storage,

UOsm significantly decreased after 1 day (mean difference -54 mmol∙kg-1, P=0.020), 2 days

(mean difference -57 mmol∙kg-1, P=0.001), and 7 days (mean difference -59 mmol∙kg-1,

P<0.001). Figures 2 presents in orthogonal diagrams with the line of identity the urine osmolality

between fresh and refrigerated urine samples stored at 7 °C (A-C) and frozen samples stored

-20°C (D-F) for each time point (1, 2, and 7 days). Both refrigerated and frozen samples were

vortexed prior to the analysis. Almost all samples point in the A-C diagrams are on the top of the

identity line indicating good agreement in urine osmolality analysis between fresh and 7°C

samples. In contrary, most samples stored in -20°C (D-F) showed a systematic decline in values

compared to fresh.
 Table 2 shows the Bland-Altman analysis for UOsm for centrifuged urine samples. In the

22 °C environment, UOsm was not different from fresh after 1 day (mean difference +5

mmol∙kg-1, P=0.225) and 2 days (mean difference +8 mmol∙kg-1, P=0.069), but it was significant

higher after 7 days (mean difference +18 mmol∙kg-1, P>0.001). In the 7 °C environment, there

were no significant differences from fresh after 1 day (mean difference +3 mmol∙kg-1, P=0.404),

2 days (mean difference +1 mmol∙kg-1, P=0.810) and 7 days (mean difference +7 mmol∙kg-1,

P=0.085). In the -20 °C storage temperature, UOsm significantly declined from fresh after 1 day

(mean difference -46 mmol∙kg-1, P>0.001), 2 days (mean difference -45 mmol∙kg-1, P>0.001),

and 7 days (mean difference -47 mmol∙kg-1, P=0.002). In the -80 °C environment, UOsm also

significantly declined from fresh after 1 day (mean difference -54 mmol∙kg-1, P>0.001) and 7

days (mean difference -22 mmol∙kg-1, P=0.005). However, there was not a statistical significant

difference from fresh after 2 days (mean difference -4 mmol∙kg-1, P=0.677). Figures 3 presents in

orthogonal diagrams with the line of identity the urine osmolality between fresh and refrigerated

urine samples stored at 7°C (A-C) and frozen samples stored at -20°C (D-F) for each time point

(1, 2, and 7 days). Both frozen and refrigerated samples were centrifuged prior to analysis. A-C

depicts the centrifuged UOsm sample data for 7°C storage for each time point (24h, 48h, and 7

days) as plotted with the line of the identity compared to fresh sample analysis. D-F represents

the sample data for -20°C storage for each time point (24h, 48h, and 7 days) as plotted with the

line of identity compared to fresh sample UOsm. Similary to the vortexed samples, almost all

samples point in the A-C diagrams are on the top of the identity line indicating agreement in

urine osmolality between fresh and 7°C samples. Just like in vortexed samples (Fig. 2) storing

urine samples in -20°C (D-F) showed a systematic decline in values compared to fresh.
 Table 3 shows the Bland-Altman values of USG for vortexed values. In the 22 °C

environment, USG was not different from fresh after 1 day (mean difference 0.000, P=0.865), 2

days (mean difference 0.000, P=0.638), and 7 days (mean difference 0.000, P=0.915). Similarly,

in the 7 °C environment, USG was not different from fresh after 1 day (mean difference 0.000,

P=0.701), 2 days (mean difference 0.000, P=0.443), and 7 days (mean difference 0.000,

P=0.644). In the -20 °C environment, USG did not differ from fresh only after 1 days (mean

difference -0.001, P=0.323), but significantly declined after 2 days (mean difference -0.002,

P<0.001) and 7 days (mean difference -0.002, P<0.001). Similarly, in the -80 °C environment,

USG valued significantly declined from fresh after 1 day (mean difference -0.002, P<0.001), 2

days (mean difference -0.002, P<0.001), and 7 days (mean difference -0.002, P<0.001).

Table 4 shows the Bland-Altman results for UC for vortexed samples. UC seemed to

significantly decrease in all storing conditions after 1, 2, and 7 days. Only vortexed values are

presented for USG and UC as there were no differences between vortexed and centrifuged

treatments for either analysis.

Multifactoral analysis comparing vortex and centrifuge methods for each time and

temperature revealed differences only in UOsm analysis. In the -20 °C storing condition, vortex

analysis after 2 days (mean difference -79 mmol∙kg-1) showed more of a decline from fresh than

centrifuge analysis for the same and temperature (mean difference -45 mmol∙kg-1; P<0.001).

Similarly, in the -20 °C storage, vortex analysis after 7 days (mean difference -86 mmol∙kg-1)

showed more of a decline from fresh than centrifuge analysis for the same and temperature

(mean difference -47 mmol∙kg-1; P <0.001). In the -80 °C environment, vortex analysis after 2

days (mean difference -57 mmol∙kg-1) showed more of a decline from fresh than centrifuge

analysis for the same and temperature (mean difference -4 mmol∙kg-1; P <0.001). Similarly, in
the -80 °C environment, vortex analysis after 7 days (mean difference -59 mmol∙kg-1) showed

more of a decline from fresh than centrifuge analysis for the same and temperature (mean

difference -22 mmol∙kg; P <0.001).

DISCUSSION

The main findings of the current investigation were: a) urinary hydration markers were

stable in room (22 °C) and refrigerated (7 °C) temperatures for up to 2 days, and up to 7 days

only in refrigerated conditions; b) urinary markers showed drastic decreases in osmolality,

specific gravity, and color when stored in either the freezer (-20°C) or deep freezer (-80°C); and

c) vortexing or centrifuging the urine sample prior to analysis after storage in both room (22 °C)

and refrigerated (7 °C) temperatures for up to 7 days did not affect the analysis. To our

knowledge, this is the first study quantifying the effects of storing temperatures and time, up to 7

days, on urinary hydration markers.

These findings confirm and expand the stability that has been previously published on

storing urine samples in cooler temperatures (5, 6). The least reliable results were obtained when

samples were stored in freezing temperatures (-20 °C and -80 °C) for up to 7 days. Even after 24

h, UOsm and USG showed a significant drop in value when stored in freezing and deep freezing

conditions. This could be due to dilution of the sample following the “freeze-thaw” method often

used in research which has been previously shown to lower urinary pH and underestimate

urinary proteins (12, 18, 25). However, there has been limited research on the effect of freeze-

thaw methods on UOsm, compared to the extensive research of freeze-thaw methods on plasma

osmolality (8, 26).

 Our data also revealed slight increases in UOsm over time, up to 7 days, when stored in

room temperature (22 °C). This is in agreement with Bezuidenhout et al. (6) who found a slight

(2 mOsm∙kg-1) increase up to 36 h. Similar trends can also be seen in refrigerated environments

after 1 day, 2 days, and 7 days, but did not reach any statistical significance. The effect of storing

samples at warmer temperatures was not the overall goal of the present study, but it shows an

interesting trend of how non-freezing conditions could increase UOsm overtime.

The effect of storage time and temperature on USG and UC has rarely been studied. Hunt

et al. (16) looked at the effects of 24 h of urine storage in both room temperature and refrigerated

environments and showed that storage temperature and time had no effect on USG. However, in

the present study, UC showed minor, although significant decreases when stored in all

environments for up to 7 days. Our data showed excellent stability of USG measurements up to 7

days in both room temperature and refrigerated environments. Previous research investigating

the effect of temperature and USG found that as temperature of the urine increased, USG

decreased (22). However, it should be noted that the urine reached room temperature prior to

being analyzed, regardless the storage environment.

Lastly, as urine samples remain stagnant, regardless of the temperature, sedimentation

develops, but no current literature addresses sedimentation of samples and what procedures

should be taken to ensure an accurate hydration assessment (1, 14). Some laboratories to address

this issue, might vortex or centrifuge the samples prior to the analysis (10). In the present study,

there was no difference in urinary hydration markers whether the samples were vortexed or

centrifuged when they were stored in either room temperature (22 °C) or refrigerated (4 °C)

environments. However, there were differences between vortex and centrifugation treatments in

UOsm when the samples were stored at -20 °C and -80 °C after 2 days. This protocol showed
that storing urine samples in freezing condition does not produce reliable results whether

samples were centrifuging or vortexing prior to the analysis.

In conclusion, the data indicated that urine specimens analyzed for UOsm and USG

remained stable in refrigerated (4 °C) environment for up to 7 days, and in room temperature (22

°C) for up to 2 days. However, UC was not stable in any storage environment following fresh

analysis. Lastly, when storing urine samples in room temperature and refrigerated environments,

there were no differences when samples were vortexed or centrifuged. However, after 2 days of

freezing and deep freezing treatments, the vortexed samples had more of a decrease from their

fresh sample values compared to centrifuged samples.

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Table 1. Bland-Altman Results: Mean difference in urine osmolality of fresh sample vs. vortexed

sample. Upper and Lower 95% CI, and P-Value.

Storing Time Mean Difference from Fresh Upper Lower

P-value
Temperature (days) (mmol∙kg )-1
95% 95%
1 +5 +14 -3 0.216
22 °C 2 +9 +18 0 0.044
7 +19 +30 +8 0.001
1 +3 +4 -12 0.467
7 °C 2 +4 +4 -13 0.349
7 +8 +17 -1 0.090
1 -50 -20 -80 0.002
-20 °C 2 -79 -50 -109 0.001
7 -86 -61 -112 0.001
1 -54 -9 -98 0.020
-80 °C 2 -57 -35 -79 0.001
7 -59 -43 -76 0.001
Table 2. Bland-Altman Results: Mean difference in urine osmolality of fresh sample vs.

centrifuged sample. Upper and Lower 95% CI, and P-Value.

Storing Time Mean Difference from Fresh Upper Lower

P-value
Temperature (days) (mmol∙kg-1) 95% 95%
1 +5 +14 -3 0.225
22°C 2 +8 +16 0 0.069
7 +18 +29 +8 0.001
1 +3 +4 -4 0.404
7°C 2 +1 +12 -9 0.810
7 +7 +4 -16 0.085
1 -46 -26 -66 0.001
-20°C 2 -45 -19 -70 0.001
7 -47 -19 -75 0.002
1 -54 -31 -78 0.001
-80°C 2 -4 +15 -23 0.677
7 -22 -7 -37 0.005
Table 3. Bland-Altman Results: Mean difference in urine specific gravity of fresh sample vs.

vortexed sample. Upper and Lower 95% CI, and P-Value.

Storing Time Lower

Mean Difference from Fresh Upper 95% P-value
Temperature (days) 95%
1 0.000 0.000 0.000 0.865
22°C 2 0.000 0.000 0.000 0.638
7 0.000 +0.001 -0.001 0.915
1 0.000 0.000 0.000 0.701
7°C 2 0.000 +0.001 0.000 0.443
7 0.000 +0.001 0.000 0.644
1 -0.001 +0.001 -0.002 0.323
-20°C 2 -0.002 -0.001 -0.003 <0.001
7 -0.002 -0.001 -0.003 <0.001
1 -0.002 -0.002 -0.003 <0.001
-80°C 2 -0.002 -0.001 -0.003 <0.001
7 -0.002 -0.001 -0.003 <0.001
Table 4. Bland-Altman Results: Mean difference in urine color of fresh sample vs. vortexed

sample. Upper and Lower 95% CI, and P-Value.

Storing Time Mean Difference from Upper Lower

P-value
Temperature (days) Fresh 95% 95%
1 -0.4 -0.2 -0.6 <0.001
22°C 2 -0.5 -0.3 -0.7 <0.001
7 -0.3 -0.1 -0.5 <0.001
1 -0.4 -0.2 -0.6 <0.001
7°C 2 -0.5 -0.3 -0.7 <0.001
7 -0.4 -0.2 -0.5 <0.001
1 -0.6 -0.4 -0.8 <0.001
-20°C 2 -0.6 -0.3 0.8 <0.001
7 -0.5 -0.3 -0.7 <0.001
1 -0.5 -0.3 -0.7 <0.001
-80°C 2 -0.6 -0.3 -0.8 <0.001
7 -0.6 -0.4 -0.8 <0.001
Figure 1
Figures 2
Figures 3
Figure Legends

Figure 1. Outline diagram for analysis of each samples

Figures 2. A-C: Values of UOsm (mmol∙kg-1) for the vortexed refrigerated (7°C) samples plotted

against the fresh samples at each time point (1, 2, and 7 days). The solid line depicts the line of

identity. D-F: Values of UOsm (mmol∙kg-1) for the vortexed frozen (-20°C) samples plotted

against the fresh samples at each time point (1, 2, and 7 days).

Figures 3. A-C: Values of UOsm (mmol∙kg-1) for the centrifuged refrigerated (7°C) samples

plotted against the fresh samples at each time point (1, 2, and 7 days). D-F: Values of UOsm

(mmol∙kg-1) for the centrifuged frozen (-20°C) samples are plotted against the fresh samples at

each time point (1, 2, and 7 days).