Clinical Chemistry Sampling and Assessment in Juvenile Rats – Reduction in and/or
Elimination of the Need for Additional Subsets of Pups
SK Clubb and L Jardine, Charles River, Tranent, Edinburgh EH33 2NE, UK
Abstract
SOT, San Diego, CA
Clinical chemistry parameters are routinely used in
adult animals and are also used to assess function
in juvenile animals. However, in juvenile animals
there is a limit to the amount of blood that can be
taken from each pup and for these studies multiple
subsets of additional animals with terminal
endpoints are required. In contrast, microsampling
has helped in the reduction of the volume of blood
samples required for TK analysis in juvenile studies
(Powles-Glover et al). To continue to reduce the
number of animals needed for these studies, the
sample volume required for clinical chemistry
parameters was assessed but limited by the volume
needed by the analyser for assessment and the
accuracy of the assay if the sample is diluted.
03/2015
In view of this sufficient sample for a full clinical
chemistry profile could not be obtained but a
selection of key parameters was chosen to reflect
clear changes over the lactation period (from
neonate to weaning), assessed key target organs
(heart, liver and kidney) and were parameters that
were suitable for quantification following dilution.
Those selected were urea, aspartate
aminotransferase, alanine aminotransferase, total
protein, albumin, globulin and AG-ratio. Electrolytes
were also analysed in non-dilute samples.
Samples were successfully collected from only 1-7
pups per group per sex (although more animals
were sampled). The samples were collected from
pups that had been dosed with water from post
natal day (PND) 5. Samples of 0.15 mL were
collected from the jugular vein on PND9, 0.3 mL
was collected on PND14 and 0.5 mL on PND21.
Body weight for these animals was also assessed.
The initial results in a limited sample set
demonstrate clear expected patterns over the time
assessed. The numbers of animals used on juvenile
studies could be reduced further if clinical pathology
samples could be taken from main study animals via
the jugular vein successfully, but further
investigation of the sampling technique and the
effects on other toxicological endpoints would be
needed.
Introduction
Juvenile study design has been a centre of focus
over the past years with concern not only to produce
a scientifically relevant study but one that also
minimises animal usage. For toxicokinetic
evaluation microsampling has been one of the key
Discussion
techniques successfully used to reduce the number
of animals required to obtain full exposure data sets
from pre-weanling pups, but sampling for clinical
pathology at this early stage, although key in
assessing target organ function at different
developmental stages, would normally consist of
terminal sampling in a number of rats. To reduce
this usage and allow for animals assigned to this
phase to be maintained on study we assessed a
reduced volume sampling technique; a technique
also being reviewed for use in adult toxicity studies.
Parameters were chosen based on previous inhouse work and work on performed on terminal
neonatal samples (Papworth and Clubb, 1995).
Parameters which had multiple isoenymes, showed
little change with time across this developmental
period or which could not be successfully diluted
and measured were not evaluated.
Analysis methods
PND
Urea (Urea):
Roche /Hitachi P Modular 800 Clinical Chemistry Analyser using Roche Test Kit.
Urease kinetic UV Assay developed from Talke H, Schubert GE. Klin Wschr
1965;43:174-175 (mmol/L)
Aspartate Aminotransferase and Alanine Aminotransferase (AST and ALT):
Roche/Hitachi P Modular 800 Clinical Chemistry Analyser using Roche Test Kit.
IFCC Method (U/L)
Albumin (Alb):
Roche/Hitachi P Modular 800 Clinical Chemistry Analyser using Roche Test Kit
Cat. No.11970909 216. Bromcresol green colorimetric assay with endpoint method.
Doumas B.T. et al 1971, Clin Chem Acta 31:87 (g/L)
Globulin (Glob):
Calculated by subtraction of the Albumin concentration from the Total Protein
concentration (g/L)
120
The samples were collected from pups that had
been dosed with water from PND5 at a dose volume
of 10ml/kg. The rat pups were held with one hand
around the thorax, restraining the forelimbs and
supporting the head; an appropriate sample for the
animals’ body weight was collected on each day.
These volumes were considered to be 0.15mL of
blood on PND9, 0.3mL blood on PND14 and 0.5 mL
blood from PND21.
Albumin Globulin Ratio (AG-R):
Calculated using Clinical Chemistry Plasma/Serum Total Protein and Albumin
Concentrations. Calculated Parameter (Albumin/(Total Protein-Albumin))
80
Males
Number of pup s
PND 9
Male
11
Female
13
PND 14
PND 21
3
6
3
6
Body weight for each pup was recorded daily.
The parameters selected were urea, aspartate and
alanine aminotransferase, total protein, albumin,
globulin and A:G ratio on all occasions; electrolytes
were also analysed in non-dilute samples on PND
14 and 21.
Samples were run on an Hitachi P Modular 800
which required a minimum sample volume of 80µL
and were diluted 1 in 3 for samples obtained on
PND9 and 1 in 2 or 1 in 3 for samples obtained on
PND14; all samples were diluted with distilled water.
The following methods were used.
10
11
12
13
14
15
16
17
18
0
19
20
21
The results of the analyses are detailed in the table
below (mean ± standard deviation).
9
Males
9
Females
14
Males
14
Females
21
Males
21
Females
AST
ALT
TP
6.3
± 1.6
77
± 15
18
±5
34
±2
6.3
± 1.2
78
±19
16
±4
34
±3
Alb
21M
140 ± 1.26
5.2 ± 0.46
101 ± 1.83
21F
140 ± 1.26
5.7 ± 0.62
101 ± 2.25
Urea
AST
ALT
PND9M
PND9F PND14M PND14F PND21M PND21F
TP
25
Alb
20
Glob
15
5
PND9M PND9F PND14M PND14F PND21M PND21F
Electrolyte levels were not measured at PND9 and
limited values were obtained at PND14. From the
data presented there was little change between
PND14 and PND21 except a slight increase in
sodium levels over this period.
AG-R
23
±2
11
±1
2.0
± 0.2
23
±1
11
±2
2.1
± 0.2
140
References
120
27
±1
10
±2
2.6
± 0.3
4.4
± 1.0
92
± 11
19
±4
37
±6
25
±3
11
±3
2.3
± 0.4
60
3.4
± 1.0
103
±9
62
± 12
46
±4
34
±3
11
±2
3.1
± 0.4
20
3.2
± 0.3
Animals can be sampled for a limited selection of
clinical chemistry parameters whilst remaining
inside ethical standards for blood sampling volumes.
Careful selection of appropriate parameters for use
on study should be considered as limited profiles
may be available. The samples may be taken from
main study animals, if following the precedent set by
microsampling, the data are used cautiously.
160
38
±1
11
±1
Conclusion
10
0
Sampling was easy and effective with a high rate of
success. The jugular was a suitable sampling site
and the analytical methods clearly detected
changes across time. Clear changes in normal
development of primarily the liver, and to some
extent the kidney and immune function, were seen
and therefore any changes to this normal
development pattern could be detected. The most
suitable parameters were urea, liver enzymes, total
protein and albumin – the lack of change in globulin
highlighting the maternal preparation of the fetus
and the maturity of this element of the immune
response post-natally. Electrolyte values as
expected were similar to those of an adult by PND
21, the limited number of samples on PND14
limiting the use of this parameter due to the sample
volume required. The high urea level reflects a low
glomerular filtration rate in the immature kidney and
immature liver function, but also a low protein and
low colloid osmotic pressure leading to low fluid
levels. Electrolytes may be effected indirectly by
osmotic pressure but also directly by decreased
reabsorpion in the neonate. Enzyme levels are low
in the immature liver immaturity but there is an
increase in enzyme levels and also in the size and
function of the hepatoblasts to weaning.
An important aspect of the success of this sampling
depends on the sensitivity of the analyser or
analytical method and hence the volume of sample
required. Further work on including more or
different samples for analysis should be
undertaken.
AG-R
Glob
35
±1
97
30
17
±3
47
±2
6.0
45
92
±12
59
±5
134
50
3.9
± 0.8
94
± 51
14F
Total protein, albumin and A:G ratios increased to
PND21, although the values between PND9 and 14
were similar and the greatest difference was
observed to PND21.
100
3.6
± 0.6
96
35
21.9 25.1 27.2 30.2 33.0 35.7 38.3 40.9 43.7 46.1 48.8 52.1 56.4 61.1
Urea
6.2
40
Females 20.5 23.8 26.2 29.2 31.9 34.7 37.1 39.8 42.4 44.8 47.3 50.7 54.7 59.5
PND
135
20
There was a increase in mean litter pup body
weight(g) from PND8-21 as presented below.
9
14M
40
Samples were successfully obtained and analysed for
all male pups and 11/13 female pups on PND9, 2/3
female pups on PND14 and all female pups on
PND21.
8
Chloride
60
Results
PND
Potassium
100
Sodium (Na), Potassium (K) and Chloride (Cl):
Roche/Hitachi P Modular 800 Clinical Chemistry Analyser using indirect Ion
Selective Electrode. Application of the Nernst equation to an electrode with crown
ether membrane type (mmol/L)
As part of an ongoing investigative study blood
samples were taken via the jugular vein from pups
on PND9, 14 and 21 for clinical chemistry
assessment.
Sodium
Urea levels were initially higher than adult animals
but declined to PND21; conversely aspartate and
alanine aminotransferase levels increased with time
from initially low levels to adult levels by the time of
weaning.
Total Protein (TP):
Roche/Hitachi P Modular 800 Clinical Chemistry Analyser using Roche Test Kit.
Biuret colorimetric assay for the formation of protein - biuret reagent complex (g/L):
Methods
Electrolytes
Na
80
K
Cl
40
0
PND9M
PND9F
PND14M PND14F PND21M PND21F
FOWLES-GLOVER, N, KIRK, S, JARDINE, L, CLUBB S. K.,
STEWART, J. Assessment of Haematological and Clinical
Pathology Effects of Blood Microsampling in Suckling and Weaned
Juvenile Rats. Regulatory Toxicology and Pharmacology, Volume
69, Issue 3, August 2014, Pages 425-433.
PAPWORTH T. A. AND CLUBB S. K. Clinical Pathology in the
Neonatal Rat. Comparative Haematology International 1995,
Volume 5, Issue 4, pp 237-250.