FUNDAMENTAL AND APPLIED TOXICOLOGY t&80-88 (1987)

Age-Related Changes in Serum Chemistry and Hematology

Values in Normal Sprague-Dawley Rats’

ST. WOLFORD, R.A.SCHROER,P.P. GALLO, F.X. GOHS,

M. BRODECK,H.B. FALK,AND R. RUHREN

Medical Research Division, American Cyanamid Company, Pearl River, New York 10965

Age-Related Changes in Serum Chemistry and Hematology Values in Normal Sprague-Daw-

ley Rats. WOLFORD, S. T., SCHROER, R. A., GALLO, P. P., GOHS, F. X., BRODECK, M., FALK,
H. B., AND RUHREN, R. (1987). Fundam. Appl. Toxieol. 8,80-88. Age-related changes in serum
chemistry and hematology values in male and female Sprague-Dawley rats 2 to 29 months of
age are described. The number of values per determination (N) ranged from 554 to 1126. Serum
chemistry parameters demonstrating changes with time included inorganic phosphorus, total
protein, cholesterol, triglycerides, and alkaline phosphatase. There was little or no sex difference
among these parameters. Hematologic parameters demonstrating changes with time included
leukocyte elements (lymphocytes, neutrophils, eosinophils, monocytes), erythrocyte parameters
(red blood cell count, hematocrit, hemoglobin, mean cell volume, mean cell hemoglobin), and
platelet count. Most trends in erythrocyte parameters were more marked in males than in fe-
males. To visualize the effect of age of these parameters, the data were plotted vs time, and
fitted models were superimposed on the graphs. Comparisons with available literature values are
made, and the value of an age-related data base when conducting long-term toxicity studies is
discussed. o 1987 society ofToxicology.

Despite wide use of the outbred laboratory rat
in toxicology studies, there are relatively few
references to normal serum chemistry and
hematology values in this species (Mitruka
and Rawnsley, 198 1; Weil, 1982; Lewi and
Marsboom, 198 1; Sanderson and Phillips,
198 1; Streett and Highman, 197 1; Caisey and
Ring, 1980; Burns and De Lannoy, 1966).
With the exception of Lewi and Marsboom
( 198 I), these investigators report values on a
limited number of parameters, a small num-
ber of animals, or without specifying animal
age. Most toxicology studies begin with
young animals, weanlings in the case of life-
time studies, but may extend into senescence.
Thus, age-related as well as treatment-related
changes may be observed in these animal
populations, making interpretation difficult
if the laboratory reference range values were
extracted from animals of unknown ages.
The purpose of this paper is to present data
demonstrating the effect of age on several se-
rum chemistry and hematology parameters.
These data were derived from a large data
base consisting of control values obtained
over a 5-year period. For those wishing to see
a more detailed description of the data base,
it should be noted that a summary paper of
the serum chemistry and hematology data
from several species has recently been pub-
lished (Wolford et al., 1986).
METHODS
Rats (Rattus norvegicus) were obtained from Charles
River Breeding Laboratories.’ The strain used was Cr I :
COBS CD (SD), which is a Sprague-Dawley rat. For this

’ Portions of this data were presented previously at the

24th Annual Meeting of the Society of Toxicology 2 Charles River Breeding Laboratories, Wilmington,
(March 1985). Mass.

0272X)590/87 $3.00 80
Copyright 0 1987 by the Society ofToxicology.
All rights of repmductioo in any form reserved.
 AGE-RELATED CHANGES IN NORMAL RATS 81

TABLE 1
SUMMARY OF AGE-RELATED CHANGES IN SERUM CHEMISTRY VALUES IN RATS 2 TO 29 MONTHS OF AGE

Corresponding
Parameter Trend observed figure number

Parameters in which a trend was observed

Inorganic phosphorus Decrease 1
Total protein Increase in first year, then decrease 2
Cholesterol Increase 3
Triglycerides Increase to -600 days, then decrease in old age 4
Alkaline phosphatase Decrease (gradual) -
Parameters in which no trend was observed
Sodium
Potassium
Chloride
Calcium
Urea nitrogen
Creatinine
Total bilirubin
Glucose
Aspartate aminotransferase
Alanine aminotransferase

compilation, control animals from pharmaceutical toxi-
cology studies were used. Animals ranged in age from 2
to 29 months. The animals were housed individually in
stainless-steel cages in temperature- and humiditycon-
trolled rooms. Twelve hours of continuous fluorescent
lighting was provided daily. Animals were fed Purina3
certified rodent chow 5002 (meal); food and water were
provided ad libitum.
Blood was collected from each conscious animal by
puncturing the venous plexus in the orbit behind the eye-
ball. Blood for serum chemistry was collected in red-top
(no anticoagulant) Vacutainefl tubes; blood for hematol-
ogy was collected in lavender-top (EDTA K,) Vacutainer
tubes.
To obtain serum, samples were allowed to remain at
room temperature for approximately 20 min. A Sep II
J? separator was placed in each tube and the tubes were
centrifuged at 2500g at 20°C for 15 min to separate the
cellular components. Serum samples were either ana-
lyzed fresh or stored at -2o’C for no longer than 28 days
prior to analyzing. An earlier study (Falk et al., 198 1) had
shown that rat serum samples could be kept under these
conditions with no apparent change in the values.
The Astra-46 was used for the determination of so-
dium, potassium, and chloride content ofthe serum. Ion-
3 Ralston Purina Company, Richmond, Ind.
4 Becton-Dickinson Division of Becton-Dickinson
and Co., Rutherford, N.J.
5 General Diagnostics Division of Warner-Lambert
Co., Morris Plains, N.J.
6 Beckman Instruments Inc., Brea, Calif.
specific electrodes were used for the determination of so-
dium and potassium; coulometry was used for chloride
determination.
A centrifugal analyzer (Centrifichem System 4OO)‘was
used to measure the following parameters in serum: urea
nitrogen (urease method), creatinine (Jaffe reaction), glu-
cose (hexokinase), inorganic phosphorus (ammonium
molybdate), cholesterol (cholesterol esterase/cholesterol
oxidase), total bilirubin (diazo reaction), total protein
(biuret reaction), alkaline phosphatase (pnitrophenyl
phosphate reaction), aspartate aminotransferase (oxalac-
etate + NADH to malate + NAD+), alanine aminotrans-
ferase (pyruvate + NADH to lactate + NAD+), calcium
(cresolphthalein complexone reaction), and triglycerides
(hydrolysis of triglycerides by microbial lipases liberating
glycerol which then reacts enzymatically to produce a
colored formazon). All tests run on the centrifugal an-
alyzer were performed at 30°C.
Red and white blood cell counts, platelet count, hema-
tocrit, hemoglobin, mean cell volume, and mean cell he-
moglobin (amount and percentage) were determined on
a Coulter S-Plus’ instrument. Percentages of reticulo-
cytes and normoblasts were read manually. The leuco-
cyte differential determinations were done using Wright
stain smears, which were read either manually or on a
Hematrak’ differential counting instrument. It was as-
certained that there was no difference between the counts
’ Baker Instruments Corp., Allentown, Pa.
* Coulter Electronics, Hialeah, Fla.
9 Geometric Data Division of Smith-Kline, Wayne,
Pa.
82 WOLFORD ET AL.

conducted manually as opposed to those conducted on Values for both parameters were higher in
the Hematrak. males than in females. An increase in total
All toxicology data (control and treated) were entered
into a DEC 10” System 1022” data base via either key protein values was observed during the first
entry or direct transmission from a VAX 1 l/785 com- year of life. Protein values leveled off, then
puter. The control data were then extracted from the Sys- were depressed in older (>2 years) animals.
tem 1022 data base. The number of data values per Cholesterol and triglyceride values increased
chemistry determination ranged from 649 to 676 for with age; however, triglyceride values de-
males and from 554 to 58 1 for females; the number per
hematology determination ranged from 1096 to 1126 for creased after approximately 600 days.
males and from 10 15 to 1030 for females. All values were There appeared to be a higher white blood
included in a regression analysis, using SAS’* proce- cell (WBC) count in both young (4 months)
dures, as follows. and old (>2 years) rats, particularly females;
It was desired to obtain a simple class of mathematical there was much more variation in the data at
models to be used for descriptive purposes to aid in visu-
alizing possible systematic changes over time. After some these points than in the middle portion of the
investigation we arrived at the following class: animal’s life. WBC values were lower in fe-
males than in males. Lymphocyte counts (ab-
Parameter =A + [BX log(age)] + (CX [log(age)]*) (1)
solute and percentages) decreased with age (2
Parameter = A + (B X age) + (C X age*) (2) months-2 years) in both sexes, while neutro-
where A, B, and C are regression coefficients to be esti- phi1 counts (absolute and percentages) in-
mated. This class seemed genera1 enough to describe the creased after 6 months to 1 year. Generally,
data; that is, for each parameter at least one of these two
eosinophil counts (absolute and percentages)
models resulted in a curve providing a good fit to the
data. The coefficients were estimated using ordinary un- were constant or showed very slight increases
weighted least squares applied to all data points (includ- with age; however, among many of the oldest
ing determinations from the same animals taken at animals these values were elevated dramati-
different time points); this was done through the general cally.
linear models procedure in SAS. Red blood cell (RBC) counts demonstrated
To visualize the data, we calculated and plotted the
mean values at each time point for which there were at a sharp increase from 2 to 3 months of age
least five measurements. The fitted models were super- (more marked in males), with a concomitant
imposed on these values, and plots of selected parameters decrease in mean cell volume and mean cell
are shown in Figs. l-9. In most cases,the trend with time hemoglobin (also more marked in males).
was similar in both sexes,and so only the figure derived Subsequently, RBC counts dropped with
from the male data is shown.
age; females had lower counts than males
throughout the lifespan. While total hemo-
RESULTS globin and hematocrit values followed the
same general course as RBC counts, the early
Tables 1 and 2 summarize the information increase was less pronounced. As RBC
regarding the parameters which demon- counts dropped, mean cell volume and mean
strated age-related changes, and indicate the cell hemoglobin increased.
corresponding figure numbers, if applicable. Platelet count declined sharply during the
All changes refer to mean values, as described first year, then leveled off or increased grad-
above. ually. Values were generally higher in males,
A decrease in inorganic phosphorus values and the early decrease was less evident in
was observed over time in both sexes, particu- males.
larly in animals less than approximately 6
months of age. Alkaline phosphatase activity DISCUSSION
declined gradually over the entire lifespan.
Serum Chemistry
lo Digital Equipment Co., Maynard, Mass.
” Software House, Cambridge, Mass. With a few notable exceptions, the age-
‘* SAS Institute, Inc., Cary, N.C. related trends observed in Sprague-Dawley
 AGE-RELATED CHANGES IN NORMAL RATS 83

TABLE 2
SUMMARYOFAGE-RELATEDCHANGESINHEMATOLOGYVALUESINRATS~TO 29 MONTHSOFAGE

Trend observed with age

Corresponding
Parameter Males Females figure number

Parameters in which a trend

was observed
White blood cells
Lymphocytes
Lymphocytes (%)
Neutrophils
Neutrophils (70)
Eosinophils
Eosinophils (%)
Monocytes
Monocytes (‘70)
Red blood cells
Hematocrit
Hemoglobin
Mean cell volume
Mean cell hemoglobin
Mean cell hemoglobin (%)
Reticulocytes (%)
Platelets
Parameters in which no trend
was observed
Basophils
Basophils (%)
Atypical lymphocytes
Atypical lymphocytes (W)
Trend was similar to that seen
in females, but less marked
Decrease
Decrease
Increase after - 1 year
Increase after -6 months
No trend until old age-more
scatter and apparent increase
then
Gradual increase-more
scatter and apparent increase
in old age
Increase
Increase
Initial increase, followed by
decrease
Minimal initial increase,
followed by decrease
Initial increase, followed by
decrease
Initial decrease, followed by
increase
Initial decrease, leveling off,
increase in old age
Very little change
Initial decrease
Trend was similar to that seen
in females. but less marked
Stabs
Stabs (%)
Normoblasts (7’0)
High initially and in old age
Decrease
Decrease
Increase after - 1 year
Increase after -6 months
No trend until old age-more
scatter and apparent increase
then
Very slight increase-more
scatter and apparent increase
in old age
Very slight increase
Increase
Initial increase, followed by
decrease-less marked than
in males
Possible initial increase,
followed by decrease
Initial increase, followed by
decrease-less marked than
in males
Trend was similar to that seen
in males, but less marked
No trend, or possibly a slight
increase in old age
Very little change
No trend
Decrease which was more
marked initially, followed by
leveling off; apparent
increase in old age

rats have been reported in other strains as 1970). However, in contrast to the extensive
well. These include decreaseswith time in study conducted by Lewi and Marsboom
serum alkaline phosphatase activity and in (198 1) in the Wistar rat, we did not observe
inorganic phosphorus values in young ani- the marked decreasesin alkaline phospha-
mals (Fig. l), generally explained by bone tase activity and inorganic phosphorus val-
growth and increased enzyme activity asso- ues between 2 and 3 months. Both parame-
ciated with it (Freedland and Kramer, ters decreasedthroughout the life of the ani-
84 WOLFORD
FIG. 1. Age-related changes in serum inorganic phos-
phorus values in normal male rats.
mals, although alkaline phosphatase de-
clined very gradually.
Total protein values increased moderately
during the first year of life in both sexes in our
study (Fig. 2) and the Wistar rat study (Lewi
and Marsboom, 198 1). However, in Long-
Evans rats, protein values did not change
with time (Kozma et al., 1969). The physio-
logical reason for this increase is not com-
pletely understood, since there was no obvi-
ous pattern of change in the albumin and
globulin concentrations in these studies. No
consistent age-related pattern in the albumin
to globulin ratio (A/G) was seen in dogs, ei-
ther (Pickrell et al., 1974; McKelvie et al.,
1966) although, based on findings in our lab-
: ..
FIG. 2. Age-related changes in serum total protein val-
ues in normal male rats.
ET AL.
FIG. 3. Age-related changes in serum cholesterol values
in normal male rats.
oratory (unpublished data), the greatest
changes in these parameters are observed be-
fore 2 to 3 months of age, and thus would not
have been seen in these studies.
As reported by other investigators studying
very small numbers of Sprague-Dawley rats
(Carlson et al., 1968; Uchida et al., 1978),
cholesterol and triglyceride levels increased
with age in both sexes (Figs. 3 and 4). In our
laboratory cholesterol values continued to in-
crease throughout the lifespan; triglyceride
values, on average, declined in old age. Lewi
and Marsboom ( 198 1) reported that, in con-
trast to the Sprague-Dawley rat, cholesterol
values in the Wistar rat increased in early life,
but leveled off at around 10 months of age
FIG. 4. Age-related changes in serum triglyceride val-
ues in normal male rats.
 AGE-RELATED CHANGES
in both sexes. Kritchevsky and Tepper ( 1964)
reported that serum cholesterol levels in BN
and Lewis inbred strains of rats decreased
between 30 and 90 days, while those of
Wistar rats increased. Story et al. (1976) re-
ported that cholesterol and triglyceride levels
were constant from 1 to 18 months in Fisher-
344 rats; both parameters increased after this
time. However, Uchida et al. ( 1978) reported
lower triglyceride values in old male Wistar
rats (approximately 2 years), possibly due to
decreased food intake. Clearly, there is a
marked difference among strains. It was
noted by Story et al. ( 1976) that lipid metabo-
lism in Fisher rats is different from that in
Sprague-Dawley and Wistar rats. Another
important consideration is the composition
of diet, which was not the same in each of the
studies discussed here.
Hematology
In contrast to serum chemistry data, there
is a considerable amount of hematological in-
formation available from the rat and mouse.
In general, as discussed below, our values for
red blood cell parameters were in agreement
with those of other laboratories, while those
for white blood cell parameters displayed nu-
merous differences.
In our study the WBC count was higher
among the young (~5 months) and old (>2
years) rats. The decrease in WBC count in
mid-life was accounted for mostly by a de-
crease in the number of lymphocytes (see be-
low), and is less marked in males because the
drop in lymphocytes is less dramatic than in
females and the number of monocytes in-
creases during this time in males. The WBC
increase in old age appears to be due to an
increase in neutrophils and eosinophils. In
contrast to our study, Lewi and Marsboom
( 198 1) reported a gradual increase in white
blood cell count in Wistar rats starting at
around 9-10 months of age, extending to 24
months of age in the male, but leveling off at
around 20 months in the female. Everitt and
Webb (1958) reported no significant change
IN NORMAL RATS 85
FIG. 5. Age-related changes in percentage of lympho-
cytes in normal male rats.
in Wistar rats from youth (about 10 months)
through senility (about 32 months) in healthy
rats. Higher counts were also observed in se-
nescent mice (Finch and Foster, 1973).
In our study, lymphocyte counts (absolute
and percentages) decreased with age through-
out the lifespan (2 months to 2 years) in both
sexes (Fig. 5). With respect to lymphocyte
percentage, our data were similar to those
seen in the Wistar rat (Lewi and Marsboom,
198 1). The trend in absolute lymphocyte
count in their study was similar to the total
white blood cell count in males, i.e., gradual
increase with age to 24 months. In female
Wistars the absolute lymphocyte count was
depressed in the middle ages (5- 15 months)
compared with earlier or later time periods.
Interestingly, a similar age- and sex-related
pattern was observed in mice (Talbot et al.,
1965). Lymphocytes were also shown to
increase in senescent mice (Finch and Fos-
ter, 1973).
The neutrophil count (absolute) was rela-
tively constant until after the first year of life,
when it began to increase moderately in both
sexes and then accelerated in old age such
that the counts eventually were equal to or
greater than lymphocyte counts. The neutro-
phi1 percentage began to increase at around
6 months (Fig. 6), which is consistent with a
concomitant drop in the percentage of lym-
phocytes. This finding is consistent with that
86 WOLFORD ET AL.

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FIG. 6. Age-related changes in percentage of neutro- FIG. 7. Age-related changes in red blood cell count in
phils in normal male rats. normal male rats.

observed in Wistar rats (Sanderson and Phil- continued to decrease. At the same time, in
lips, 198 1). both sexes there was an increase in the mean
In our study, eosinophil counts (absolute cell hemoglobin (less marked in females) and
and percentages) were increased in old age. mean cell volume which is consistent with
By contrast, Everitt and Webb (1958) re- fewer and larger RBCs. The mean cell hemo-
ported marked decreases in eosinophil counts globin concentration (percentage) was rela-
with age, especially in the last 50 days of life, tively constant in males and decreased
a phenomenon which is also seen in terminal slightly in females. By contrast, Everitt and
disease in man and is attributed to adrenal Webb (1958) reported increased red blood
cortex stimulation (Jennings, 1952). This cell count as well as hemoglobin in old male
trend was not observed, however, in the large Wistar rats. They reported that this finding
Wistar rat study (Lewi and Marsboom, 198 1) occurred in the last stages of life, regardless of
nor in old or senescent mice (Talbot et al., the actual age. Other investigators, however,
1965; Finch and Foster, 1973). In our study have reported decreases in older Wistar rats,
monocyte percentages increased with time. and so it is difficult to ascertain if the effects
Lewi and Marsboom ( 198 1) also reported an reported were due to age alone, to underlying
increase in monocyte percentages between disease, or to both.
approximately 2 and 18 months followed by
a leveling off through 24 months. 58
1
In our data, RBC counts increased between
the ages of approximately 2 and 3 months
and, because the increases in hematocrit and
hemoglobin values were less marked, there
was a concomitant decrease in mean cell vol-
ume and mean cell hemoglobin (Figs. 7 and
8). These trends were more apparent in males
than in females. Later in life RBC count as
well as hemoglobin and hematocrit appeared
to decrease, while mean cell volume and
mean cell hemoglobin increased. Similar
changes were observed in both sexes of young
Wistar rats (Lewi and Marsboom, 198 1). In FIG. 8. Age-related changes in mean cell volume in
old age, our RBC and hemoglobin values normal male rats.
 AGE-RELATED
7oo;j---y -7-p’ r--1
WI3
Age. days
FIG. 9. (A) Age-related changes in platelet count in normal male rats. (B) Age-related changes in platelet
count in normal female rats.
A drop in platelet count during the first
several months of life was followed by a grad-
ual increase (males) or a leveling off (females)
(Fig. 9). Interestingly, the Wistar rat data pro-
vided a similar shape of curve, but the de-
crease occurred much earlier in life (Lewi and
Marsboom, 198 1). By 3 months of age, the
male rat values were relatively constant, and
by 5 months the female rat values were con-
stant.
In conclusion, we have presented a body of
data which comprises a sizable portion of the
available reference information on Sprague-
Dawley rats. To date we are unaware of any
data base which is as large or as complete with
respect to ages or number of parameters. It
should be emphasized that the collection of
such data is worthwhile only if it is put to use
on a regular basis. To that end, we have de-
rived “reference ranges” for several age
groups, consisting of all values falling be-
tween the 10th and 90th percentiles of these
normal values. Based on visual examination
of the data presented here, we have chosen
three characteristic age groups for the rat: less
than 6 months, 6 to 18 months, and greater
than 18 months. While it would be possible
theoretically to subdivide the data into more
age groups, restricting the number of groups
to three allows us to store and quickly search
for the reference range values for each param-
eter. Every study value obtained in our labo-
CHANGES IN NORMAL RATS 87
1100 1000
Age. days
ratory is compared with the reference range
for the corresponding species, sex, and age
group. If it is outside this range, it automati-
cally is flagged so that it can be identified
quickly.
Thus the data presented here have been put
to practical use by helping us to screen large
data sets rapidly and ultimately to make deci-
sions about the compound under investi-
gation.
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