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This is to certify that the

thesis entitled

'D is‘l'rlbidtoncrwan‘l'otheni c. Act A
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Date Aug. 8, 1989

 

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(18555 5 c8

DISTRIBUTION OF PANTOTHENIC ACID IN THE RAT

BY

Dana I. Wu

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1989

ABSTRACT
DISTRIBUTION OF PANTOTHENIC ACID IN THE RAT
BY

Dana I. Wu

Forty-eight male Sprague-Dawley weanling rats were used

to determine the distribution of pantothenic acid (PA) in
organs, blood and urine. The purpose was to determine the
best indicator of PA status by correlating PA concentration
of bodily fluid with those of organs. The rats were divided
into 4 groups and fed PA deficient AIN-76 diets supplemented
with: A)120 mg Ca-PA/kg diet, ad lib: B)12 mg/kg, ad lib;
C)O mg/kg, ad lib; D)12 mg/kg, pair-fed with group C for

8 weeks. Heart, spleen, kidney, whole blood, plasma and
urine were collected at week 2, 5 and 8 for determination

of total and free forms of PA. Free PA concentration in
kidney and total PA concentration in heart, spleen and whole
blood of group C were significantly lower (p<0.05) than
those of group B at all weeks examined. Free PA
concentration in plasma correlated the best with heart,
spleen, kidney free PA concentration in group C and was

identified to be the best indicator of PA status.

ACKNOWLEDGEMENTS

My sincere thanks Dr. Won Song for her guidance and support
throughout my graduate program. Thanks are also extended
to my committee members: Dr. M. Bennink, Dr. J. Bond, and
Dr. J. Gill for all their advice and assistance. To my
"lab-mates": C. Bates, K. Ellerman, D. Gould, S. Nied,

M. Schafer-Greely, and M. Schneider for their valuable
friendship. To Dr. Maureen Storey who has been a real
inspiration to me. To my loving parents whose love and
support could be felt across the miles. Lastly, my deepest
thanks to my husband, Robert Wassmer, for his unconditional
love, encouragement and faith in me.

iii

TABLE OF CONTENTS

List of Tables.. ......................... . ............... vi
List of Figures. ......................................... Vii
Introduction... ...... .... ............................. ...1
Review of Literature ..................................... 4
Discovery.......... .............................. .....4
Analytical Methods...................... ........ . ..... 5
Pantothenate Requirement ......... ... ........ ... ....... 7

Pantothenic Acid Deficiency: Effect on Histology of
Tissues ..................... . ...... ...................12

Pantothenic Acid Deficiency: Pantothenate Content in
Tissues, Organs and Blood.............................13

Dietary Sources of Pantothenic Acid: A Need For
concernOOOOOOO...O....OO.............OOOOOOOOOOOOOOOO.21

Intestinal Microbial Synthesis of Pantothenic Acid....22

Absorption of Pantothenic Acid ....... ..... ............ 23
Synthesis of CoA........... .............. .............26
Functions of CoA............ .......................... 27
Toxicity of Pantothenic Acid.. ........... ........ ..... 28
Materials and Methods..... ....... .... ........... .........29
Preliminary Experiments ............................... 29
Research Design............. ......... .................31
Data Collection.................. ..................... 33
Collection and Treatment of Organs ........ . ..... ......34
Pantothenic Acid Determination............. ........... 38
Statistical Analysis ......................... . ........ 39

iv

ResultSOOOOOOOOOOOO ......... 00...... ....... 09.0.00000000041

Discussion...... ......... . ............................... 72
Conclusion ........ .... .......... .... ........... ..........77
Limitation...... ................. ....... .......... .......79
Recommendation.. ..... .... ........ .................. ...... 81
Appendices

A. Food Intake of Rats................................82

B.

C.

D.

E.

F.

G.

H.

I.

weight Gain 0f RatSOOOOOOOOOOOO00.0.00000000000000083

Pantothenate (PA)
Pantothenate (PA)
Pantothenate (PA)
Pantothenate (PA)
Free Pantothenate

Free Pantothenate

Concentration
Concentration
Concentration
Concentration
Concentration

Concentration

of
of
of
of
of

in

Rat Heart.......84
Rat Spleen ...... 86
Rat Kidney......8a
Rat Whole Blood.90
Rat P1asma......92

Urine...........o3

Organ Weights of Rats..............................94

List of References. ..... . ................... .............96

Table 1:

Table 1a:
Table 1b:

Table 2:

Table 3:

Table 4:
Table 5:
Table 6:

Table 7:

Table 8:
Table 9:
Table 10:

Table 11:

LIST OF TABLES

Purified pantothenate deficient basal diet

composition ................................... .32
AIM-76TH mineral mixture ....................... 32
AIN-76TM vitamin mixture ....... . ..... ..... ..... 32

Total weight of organs and percent body weight

of organs of rats in the study ....... ..........,45

The ratio of free (F) to total (T) pantothenate
in heart, spleen and kidney of rats in the
studYOOOOOO......OOOOOOOOOOO... ..... 0.0.0.0....47
Pantothenate content in whole rat heart ........ 50
Pantothenate content in whole rat spleen.......54

Pantothenate content in whole rat kidney.......57

Correlation results of organ pantothenate (PA)
concentration vs bodily fluid of group C

rats (r)......... ...... ... ..... ........ ........ 64
Residual results of predictive equation 1......66
Residual results of predictive equation 2 ...... 68
Residual results of predictive equation 3. ..... 69

Residual results of predictive equation 4......71

vi

Figure 1:

Figure 2:

Figure 3:

Figure 4:
Figure 5:
Figure 6:

Figure 7:

Figure 8:

Figure 9:

Figure 10:

Figure 11:

LIST OF FIGURES

Structure of CoA and its derivatives..........6

Reported results of pantothenate concentration
in rat tissues and organs.....................15

Pantothenate content in cortex, medulla, and
pelVis Of rat kidney-0.0.0.0...0.0.0.00000000036

Diet intake of rats...........................43
Growth pattern of rats........................44
Concentration of pantothenate in rat heart....49

Concentration of pantothenate in rat
spleen........................................52

Concentration of pantothenate in rat
kidneYOOOOOOOOOOOOOO......OOOOOOIOOOOOOOOO....55

Concentration of pantothenate in rat whole
b100d0000000000 ..... OI.....OOOOOOOOOOOOOOOO...58

Concentration of pantothenate in rat
plasma...........O.........OOOOOOOOOOOOOOOOOOO60

Concentration of pantothenate in rat

urineOOOCCOOOOIO......OOOOOOOOOOOOOOOO00......61

vii

INTRODUCTION

It has been well established that pantothenic acid
is essential for humans and animals. Pantothenate, a B
vitamin sometimes called vitamin 8-5, is a precursor for
coenzyme A (CoA) and phosphopantetheine of acyl carrier
protein. CoA and phosphopantetheine are known to be
biochemically active and bound forms of pantothenic acid
and are required in many different metabolic reactions
(Krehl, 1954; Lehninger, 1982).

Pantothenate is found in many foodstuffs. The most
concentrated sources are egg, liver, meat, and legumes.

An average American diet has been reported to provide 2 -

3 mg pantothenate/1000 kcal (Essenstat et a1., 1986; Song
et a1., 1985: Srinivasan et a1., 1981: Walsh et a1., 1981).
Because pantothenate is widely distributed and no overt
clinical deficiency of the nutrient has been reported in
humans, it has been generally assumed that intake is
adequate for persons consuming a normal diet.

A Recommended Dietary Allowance has not been
established for pantothenic acid. However, the Food and
Nutrition Board of the National Academy of Sciences,
National Research Council has recommended an Estimated Safe
and Adequate Daily Dietary Intake of pantothenate for adult
of 4 - 7 mg/day. A Recommended Dietary Allowance will be

1

2
set after sufficient experimental data accumulate and
accurate methods of assessment are found. Studies with
experimental animal demonstrate that pantothenic acid
deficiencies can occur. Rats deficient in pantothenate
were reported to be weak and anorexic, and grew poorly
compared to normal rats. The need for pantothenate was
more evident during growth (Henderson et al., 1941:
Lotspeich, 1950; Reibel et al., 1982).

Irregular and unusual eating habits may also place
a person at risk for pantothenic acid deficiency (Eissenstat
et al., 1986). However, most of the symptoms such as
irritability and anorexia noted in humans were reversible
with the administration of pantothenic acid (Henderson et
a1., 1942).

Determining the concentration of pantothenic acid in
organs of rats fed diets deficient in or supplemented with
various amounts of pantothenic acid may give an insight
to the pattern of depletion or storage in the whole
organism. It was hypothesized in this study that 1)
distribution of pantothenic acid among various organs
differs for rats fed different amount of pantothenate 2)
pantothenic acid level in urine and blood (plasma or whole
blood) reflect pantothenic acid levels in diet and various
organs at different stages of pantothenate status. Growth
patterns and clinical symptoms also were expected to

correspond to pantothenate intake and organ level.

3

In this study, pantothenic acid levels in urine, plasma
and whole blood were measured and correlated to the
pantothenate concentration in selected organs to identify
the most valid and reliable indicator of pantothenate status
in rats. Growth patterns and clinical symptoms were also
examined throughout the study. Specific objectives of the
study included:
l.To determine the distribution of pantothenate in heart,
spleen, and kidney of rats fed deficient, adequate or
excessive amount of pantothenic acid.
2.To determine pantothenate levels in urine and blood of
rats fed deficient, adequate and excessive amount of
pantothenate for different periods.
3.To correlate pantothenic acid levels in urine and blood
with those in various organs at different stages of
pantothenic acid deficiency.
4.To examine clinical symptoms and growth patterns of rats

fed deficient, adequate and excessive amount of
pantothenate.

REVIEW OF LITERATURE

DISCOVERY

Pantothenic acid was first identified by Roger Williams
in 1933. Williams detected pantothenate as an acidic
substance which was a required growth factor for yeast
(Williams, 1943). Pantothenate was found in all plant and
animal tissues as well as in microorganisms analyzed. Thus,
Williams named it pantothenic acid ("pan-" meaning
"everywhere").

Pantothenic acid was also called "filtrate factor"
because it was not retained on the absorbent fuller earth.
Lepkovsky et al. (1936) determined that this filtrate
factor, distinct from thiamine, riboflavin and pyridoxine,
was required by rats for normal growth.

Pantothenic acid was also known as "chick
antidermatitis factor" which was discovered independently
by Woolley et a1. (1939) and Jukes (1939). The factor
prevented and cured a characteristic dermatitis in chicks
but not in rats. They described the disease as
characterized by skin lesions that could be alleviated by
administration of pantothenic acid. Thus, a need for the
status of pantothenic acid as a vitamin for animals was
established. To further understand this phenomenon, the

4

5

structure of the factor was studied. Woolley et a1. (1939)
showed that the structure was a hydroxy acid destroyed by
alkali and had a beta—alanine united through its amino group
to the carboxyl of the hydroxy acid (Figure 1).

It was not until a dozen years after Williams'
discovery of pantothenic acid that the biochemical role
of pantothenic acid was discovered by Lipmann and Kaplan
(1948). They discovered a heat-stable co-factor necessary
for promoting ATP-dependent enzymes which functions as an
acyl acceptor for the pyruvate and alpha-ketoglutarate
dehydrogenase complexes. This co-factor was later named
coenzyme A (CoA) for acetylation. Upon purification and
analysis of CoA, they discovered that it contained

pantothenate in a bound form.

ANALYTICAL METHODS

Concerns regarding the requirement of pantothenic acid
in man led to the development of several methods for
measuring pantothenate. Today, there are two commonly used
methods to measure pantothenic acid: microbiological assay
and radioimmunoassay (RIA). Microbiological assay employs
the use of a microorganism, most commonly Lagtgbagillus
plantazgm, which requires pantothenic acid for growth
(Strong, 1941). Suspensions of samples are incubated with
the microorganism and the acid produced by the organism
is a measure of pantothenate in the sample by direct

titration of entire culture. An RIA for pantothenate acid}

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was developed by Wyse et a1. (1979). RIA is based on the
binding specificity of an antibody specific for pantothenic
acid. RIA employs the use of expensive radiolabelled
chemicals; however, more samples can be assayed within a
given time when compared to microbiological methods. RIA
measures the ability of the unlabelled antigen
(pantothenate) to inhibit binding of the radioactive antigen
by antibody (rabbit antiserum). The process is a simple
competition in which antigen occupies a portion of the
antibody binding sites reducing the free antigen available
to the labelled antigen. Thus the pantothenic acid
concentration of an unknown sample is determined by
comparing the decreased labelled pantothenate binding it
produces with a standard curve. However both methods can
only measure free pantothenate. With enzymatic treatment,
bound pantothenate can be liberated and therefore be
measured for total amount of the free pantothenic acid
content. Thus, bound pantothenic acid can be determine
mathematically by simple subtraction of free pantothenate

content from total pantothenate content.

PANTOTHENATE REQUIREMENT

Rats deficient in pantothenate exhibit reduced growth
rate, loss of body weight, roughening and achromotrichia
of the coat, muscle weakness, impaired adrenal function,

inhibition of antibody formation, neural damage and

8
eventual death (Reibel et a1., 1982). Hatano (1962)
reported that weight loss and fur changes appeared only
when urinary pantothenate excretion reached 1 ug/day
compared to the control group's excretion of 95 - 118
ug/day. It took 18 days for weanling rats maintained on
a deficient diet to reach this low level (of 1 ug/day) in
urine.

Anorexia was observed by McCarthy et al. (1966) within
the first week in mink kits fed a pantothenic acid deficient
diet. Weight loss in these animals was noted consistently
after the first week. The average weight loss was 40 -
60g/week and by week 8, fifty percent of the animals had
died. The final body weight of the surviving animals at
week 8 was half their original body weight. Other signs
reported by McCarthy et a1. included emaciation,
dehydration, vomiting after water or food ingestion, and
loose mucoid-like feces which changed to melena 6 - 9 days
prior to death.

In human studies, pantothenate deficiency was
established with use of an antagonist, omega-methyl-
pantothenic acid along with a pantothenate deficient diet
(Lubin et al., 1956: Hodges et al., 1958). Omega-methyl-
pantothenic acid hastened the development of pantothenate
deficiency and increased the severity of the abnormalities.
Hodges et a1. (1958) worked with three groups of human
subjects: one group was the control and received 20 mg

of Ca-pantothenate a day, the second group received diets

9
devoid of pantothenate, and the last group received diets
devoid of pantothenate plus 750 mg - 1000 mg per day of
omega-methyl-pantothenate. Subjects remained on the diet
for nine weeks. In the third group, individuals exhibited
personality changes, complained of fatigue, sleep
disturbances and neurological disorders such as numbness,
paresthesis and muscle cramps. Whereas, the other two
groups reported no symptoms. Similarly, Lubin et al. (1956)
reported in a study in which human subjects received a diet
devoid of pantothenic acid plus 500 mg of omega-methyl-
pantothenic acid per day for 4 weeks experienced depression,
paresthesis, cardiovascular instability, neuralmotor
disorder, muscle weakness, abdominal pains and frequent
infections.

Pantothenic acid has a growth promoting effect
independent of its role through CoA (Reibel et a1., 1982;
Henderson et al., 1942: Lotspeich, 1950). Reibel et al.
reported that male Sprague-Dawley rats weighing 250 - 300
grams maintained on a pantothenic acid deficient diet did
not grow as well as the control at week four of the
experiment. These deficient adult rats, however, had
maintained CoA concentration at normal levels although free
pantothenate levels were depressed in heart, kidney,
gastrocnemius, liver, and testes at the fourth week of their
experiment.

Lotspeich (1950) also demonstrated that rats fed a

10
pantothenic acid deficient diet produced a "plateaued"
effect on weight curves in adult female Sprague-Dawley
rats when subjected to growth hormone injection as early
as the second week on a deficient diet. Under normal
conditions, a deficiency of pantothenate is reported to
be difficult to produce in adult rats. A dramatic weight
gain resulted after an intraperitoneal injection of 1.0
mg of Ca-pantothenate per day for four days into the rats
(Lotspeich, 1950).

The role of pantothenic acid to growth was further
tested by Krehl (1954). He noted that as rats matured and
reached adulthood, the requirement for pantothenate was
greatly reduced. He demonstrated this by placing young
hypophysectomized rats on pantothenic acid deficient diet.
Doing so, the rats were deprived of growth hormone as well
as other trophic hormones. These animals did not show any
sign of deficiency. In fact they remained "immature, non-
growing rats". Thus, when the impetus to grow was removed,
the requirement for the vitamin decreased. This could also
explain why it is difficult to produce a deficient state
in adult animals.

Henderson and co-workers (1942) were able to raise
four generations of rats using ration 789 (containing all
known vitamins including 20 mg Ca-pantothenate/kg diet)
without difficulty. These rats reached maturity without
optimum growth but were able to reproduce. Therefore,

Henderson et al. (1942) concluded that the requirement for

ll
pantothenic acid may be lower than the 20 mg Ca-
pantothenate/kg diet supplemented to 789 ration. In
addition, Henderson et a1. (1942) suggested that the
requirement for pantothenic acid may be lower when the diet
included all the other essential vitamins.

Pantothenate was also reported to prevent graying in
pigmented rats. In another study by Henderson et a1. (1942)
black rats, 21 days old, fed a pantothenic acid deficient
diet became gray in 4 - 6 weeks. A daily oral supplemention
of 40 ug per day was sufficient to prevent or cure
achromotrichia in most of the rats. Others report higher
levels, 80 - 500 ug, of Ca-pantothenate to prevent
achromotrichia (Unna, 1940: Ansbacher et a1., 1941). A
ruffling and rusty discoloration of albino rats were
reported as early as second week when 13 rats were fed a
deficient diet (Hatano, 1962). Prevention of pigment loss
in deficient rats was also possible if the rats were
adrenalectomized (Ralli et a1., 1944). Hair growth and
melanin deposition were suppressed in the adrenalectomized
rats when rats were injected with deoxycorticosterone
acetate. Dumm et a1. (1952) support this concept.
Hypophysectomy produced an atrophy of the adrenal cortex
and was associated with an increase in pigmentation or
decrease in graying for black rats.

Pantothenate alone, however, may not be sufficient
in curing graying. Lunde and Kringstad (1939) reported

that pantothenic acid was distinct from the "anti-gray hair

12
factor" since pantothenate alone was not effective against
graying. Oleson et a1. (1939) agreed with this
theory and suggested that a "second filtrate factor" was
necessary for normal hair color but did not specify what

the second factor was.

PANTOTHENIC ACID DEFICIENCY: EFFECTS ON HISTOLOGY OF
TISSUES

Hatano (1962) investigated the effects of pantothenate
deficiency on the histology of various organs with male
Winstar rats (weighing an average of 45 9) starting at the
age of 28 days. The liver of the rats on a deficient diet
(<8 ng pantothenate/kg diet) for 32 days had areas of
swollen cells which was not due to fatty infiltration.

The glycogen content in liver was also reported to be
decreased, though values were not reported by Hantano.
These histological findings in liver of deficient rats were
similar to those seen in riboflavin and pyridoxine
deficiency. Thus, it will be inaccurate to identify
pantothenate deficiency with these histological changes
alone. Hatano did not examine if the changes were
reversible when deficiency was not severe. The lungs of
"most all" (n=4) deficient rats in Hatano's study were
characterized by inflammation similar to prolonged
bronchopneumonia, pulmonary alveolitis, and bronchitis.

The adrenal glands showed irregular cellular arrangement

of the zona fasciculate in the deficient group. The adrenal

cortex appeared to be hyperactive. Contrarily, Deane and

13

McKibbin (1946) reported that deficient rats had adrenal
depleted of ketosteroid material and also appeared enlarged
and solid black due to hemorrhage. In addition, the adrenal
cortex was characterized by loss of lipid. The signs of
adrenal damage were magnified with the administration of
adrenocorticotrophic hormone (ACTH) in the deficient animals
(Winters et al., 1952). Cortisone appears to have a
protective influence on the adrenal. With cortisone
treatment, the adrenal of the deficient rats were
indistinguishable from those of the normal control rats.

Hatano (1962) also reported that there was a primary
decrease in spermatogenesis in the pantothenic acid
deficient rats. The deficiency group exhibited atrophic
seminiferous tubules and reduced number of seminal
epithelia. Hatano demonstrated that spermatogenesis can
be repaired, within 2 weeks, in the pantothenate deficient
rats by adding pantothenate in the diet (feeding a control
diet of 40 mg pantothenate/kg diet). Other tissues/organs
affected by a pantothenate deficiency were also reported
by others. McCarthy et a1. (1966) observed stomach
ulceration and hemorrhage in minks on a deficient diet for

eight weeks.

PANTOTHENIC ACID DEFICIENCY: PANTOTHENATE CONTENT IN
TISSUES, ORGANS AND BLOOD

The distribution of pantothenate has also been examined
by Hatano (1962) to understand pantothenate metabolism.

He determined the total and free pantothenic acid content

14

in liver, kidney, Lung, spleen, brain, heart, testes,
thymus, and whole blood of young (28 day old) male rats
by microbiological assay method using Lagtgbagillus
arabingsus (Figure 2). Total pantothenate was determined
by breaking down the bound forms to the free form by
enzymatic treatment with Lipmann’s original enzymatic
solution and then assayed for total free pantothenate.
Lipmann's original enzymatic solution consisted of pigeon
liver extract, 2% intestinal phosphatase solution and 1
M Tris hydroxymethylaminomethane (pH 8.3) mixed in a
proportion of 2:1:1.

In Hatano's study (1962) 28 day old rats (4 rats per
group) were fed either a pantothenate deficient diet or
a control diet containing 40 mg pantothenate/kg diet for
32 days. Pantothenate deficient basal diet used in Hatano's
study was a purified diet containing 67.8% sucrose, 17.0%
casein and less than 8 ng pantothenate per kg of diet. It
was noted in both groups that the highest total pantothenate
concentration, in ug/g tissue, was found in the liver.
Kidney, lung, and adrenal contained the next highest
concentration of the vitamin. The rats fed the pantothenate
deficient diet for 4.5 weeks showed a 94% reduction (37.5
ng/ml) from the control value of 615 ng/ml of pantothenate
in the blood. Other reduction of pantothenate was also
noted in lung, adrenal, testes, heart, and liver. Urinary
pantothenate values drOpped markedly from 95 - 118 ug

pantothenate per day to 1 ug per day within 18 days fed

PA concentration (ug PA/o tissue)
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Liver Kldnoy Lung Spleen Brain Heart Testis AdrenalThymueWh bid

Tissues/Organs Examined

- on wk 2.5.7 ......3 Oct wk 2.5.: : 00! wk 4.5.1 Dot wk 4.5.r
Norm wk 4.6.1': Norm wt: 4.5.- Norm wk 5.5.7353 Norm wk 6.6.F

 

Figure 2. Reported results of pantothenate concentration
in rat tissues and organs.

Data from Hatano (1962). Total (T) and free (F)
pantothenate concentration of rats fed deficient
pantothenate diet (def gp) at 2.5 and 4.5 weeks and normal
diet (+40mg Ca-Pantothenate/kg diet) for 4.5 weeks and 6.5
weeks. Values represent mean (std dev. not reported) for

4 rats per group. # Whole blood (wh bld) values reported
for norm rats at wk 4.5 are T: 615 ng/ml, F: 550 ng/ml:

at wk 6.5 are T: 650 ng/ml, F: 590 ng/ml.

16
the deficient diet. Advanced changes in the deficient
group, such as significant weight loss when compared to
the control group, were more evident by the end of five
weeks (Hatano, 1962). Minks fed a pantothenate deficient
diet for eight weeks appeared to be absent of fat especially
in the coronary grove and kidney pelvis (McCarthy et al.,
1966).

Since pantothenate is the precursor of CoA, it seems
logical to assume that the level of one will correlate with
the level of the other. However, this may not necessarily
be the case. Tissue levels of CoA were found to be lower
in weanling animals on a pantothenate deficient diet than
adult rats on the same diet (Robinsharo and Needly, 1985).
Adult rats fed a pantothenic acid deficient diet for 8 weeks
had a reduced free pantothenate content by as much as 90%
in the heart and 70% in the liver without ever affecting
the level of CoA in these tissue. It may be possible that
the need for pantothenic acid is decreased with adult
animals because of slow growth and/or a larger pool of CoA.
In young growing animals, tissue stores of pantothenate
may not be adequate to support the high rate of CoA
synthesis needed for development and therefore CoA level
is affected along with free pantothenate level. Unna and
Richards (1942) reported that the daily pantothenate
maintenance requirement of the rats decreased from 100 ug
at 3 weeks of age to 25 ug at 10 weeks. Maintenance of

CoA level with simultaneously low pantothenate in the tissue

17
level during a deficient intake of pantothenic acid
indicates that mechanisms other than dietary supply of free
pantothenate may be responsible for control of CoA levels
in mature animals. Karasawa et al. (1972) demonstrated
that an excess intake of pantothenic acid (amount and
duration of feeding not specified) did not increase the
concentration of CoA in rat kidney or liver. However, the
level of free pantothenic acid increased greatly (amounts
not reported) in the kidney and liver.

Robinsharo and Neely (1985) reported that normal rats
have the highest CoA contents in cardiac muscle and liver.
Kidney, adrenal, and white skeletal muscle followed in
concentration. The level of CoA in tissues in adult rats
remained fairly constant.

In the study of Reibel et a1. (1982), adult rats (250
300 grams) fed a pantothenate deficient diet for
four week showed that the heart, kidney, gastrocnemius,
diaphragm, adrenals, and testes contained only about 10%
of the normal level of free pantothenate, and liver
contained close to 30%. CoA content of the tissues and
organs, however, was fairly similar between the deficient
and normal groups for 8 weeks regardless of free
pantothenate levels in tissues/organs. In addition, the
level of free pantothenate at week eight was not
significantly different from the values obtained at week
four. Reibel et al. also examined if it was possible to

increase CoA synthesis by fasting or inducing diabetes when

18
glucose was not available as the energy source. They used
pantothenic acid deficient rats (consuming < 1 mg/kg diet)
containing 10% of normal pantothenate in the heart and 30%
in the liver as well as rats fed the control diet
(supplemented with 50 mg/kg diet). They reported that heart
and liver CoA content did increase in rats of both groups
examined after a 48 hour fast or an injection with 60 mg
alloxan/kg body weight to induce diabetes. They also
reported that the pantothenate deficient rats were more
responsive to the fast as well as diabetes than the control
rats (an increase of 11-13 nmol pantothenate/g dry kidney
tissue of deficient rats versus 10 nmol in the control).
To determine the source of pantothenic acid for CoA
synthesis, they examined kidney, gastrocnemius, testis,
diaphragm, adrenal and serum of the rats fed the deficient
and control diet. Pantothenic acid and CoA content
increased in organs examined in the deficient group when
fasted or diabetic. Whereas, no change in pantothenate
or CoA except free pantothenic acid level in the kidney
(p<0.001) was observed in the control rats. Thus, the
source of pantothenic acid for the elevated CoA synthesis
in the deficient pantothenate rats was not determined and
could very well have come from a source not measured by
the group. In addition, they did not specify housing
condition and the possibility of coprophagia. Due to

intestinal microbial synthesis of pantothenate, the practice

19
of coprophagy may result in extra source of pantothenic
acid from feces.

Branca et a1. (1984) demonstrated that pantetheine,
not pantothenate, increase the total CoA content in the
liver of 300 - 3509 male Wistar rats. Liver perfusion was
achieved by cannulation of the caval vein through the right
atrium and the portal vein. Perfusion with one hundred
uM pantetheine in perfusion medium increase CoA content
by 20 - 30 percent. However, less than 100 uM of
pantetheine did not increase CoA in the perfused medium.

On the other hand, 100 uM of pantothenic acid in the same
condition did not increase CoA content in liver.

Blood level of pantothenate in deficient rats showed
significantly low level (Hatano, 1962). Hatano reported
that feeding 28 day old rats with pantothenate deficient
diet resulted in a reduction in blood value (43 ng total
pantothenate/ml of whole blood) as early as the 18th day
of the experiment. The blood value remained low for 32
days of the study: a reduction of 94% in total and 97% in
the free pantothenate in the deficient group as compared
to the control (37.5 ng total/ml and 15 ng free
pantothenate/ml whole blood in the deficient rats versus
normal values of 615 ng total and 550 ng free
pantothenate/m1 whole blood). When the deficient rats were
supplemented, after 32 days, with 40 mg Ca-pantothenate/kg
diet for 14 days, free pantothenate value in blood increased

to level similar to that of the control (590 ng/ml compared

20
to the control of 550 ng/ml). However, the concentration
of bound form of pantothenate was still reduced to a ratio
of approximately 60% of the normal levels with no increase
observed for the same period of supplementation. Hatano
speculated that the reason for the bound form of
pantothenate remaining low while the free form was similar
to control rats’ may be due to the heavy demand imposed
on the bound (and active) form by various organs (Hatano,
1962).

Due to the wide variation cited in literature,
controversy exists on the normal level of pantothenate in
whole blood. Because of methodological difficulties and
differences in microorganisms employed in the assay, the
values for blood level of pantothenate for man range from
59 ng/ ml to 2622 ng/ml whole blood and 137 ng/ml to 1830
ng/ml for serum (Wyse et a1., 1985). However, fasting whole
blood and erythrocytes correlated significantly (p<0.001
using Pearson's correlation coefficients) with diet intake
(r=0.38, 0.60 respectively) (Eissenstat et al., 1986).
Only total pantothenate was determined in whole blood by
the group, thus free pantothenate data was not available.
Pearson (1941) has reported that pantothenic acid occurs
in fairly equal concentration between plasma and cells in
human. However the free form of pantothenate distribution
varies with species. For instance, horse, human, rabbit
and sheep was reported by Pearson as having a greater

concentration of free pantothenate in the cells than plasma.

21
On the other hand, the opposite was observed in dogs and
pigs. Pearson’s study (1941) with rabbits showed a ratio
of 1:1:1 in the distribution of free pantothenate in whole
blood, plasma, and pack cells of controls. However, Pearson
did not report any pantothenate blood values for rats which
makes comparison of ratio to this study using rats
difficult. Ono et a1. (1974) reported that free
pantothenate in rat blood comprised about 80% of total

pantothenate.

DIETARY SOURCES OF PANTOTHENIC ACID: A NEED FOR CONCERN
Although pantothenate is available from many food
sources, there may be a need for concern regarding
consumption of enough pantothenate to meet the Estimated
Safe and Adequate Daily Dietary Intake of 4 - 7 mg/day.
Milk, chicken, potatoes, oats, tomato, whole grains are
just a few of the items rich in pantothenate (Wyse et al.,
1985). However, processed foods, foods made from refined
grains, fruit products and fat contain low amount of the
vitamin (Walsh et a1., 1981). Eissenstat et a1.
(1986) calculated the dietary pantothenate intake from 4-
day diet records of 63 adolescents. They reported 49% of
the females and 15% of the males consumed less than 4
mg/day. The authors explained that the inadequate
consumption was due to the poor food selection of the

adolescents.

22

INTESTINAL MICROBIAL SYNTHESIS OF PANTOTHENIC ACID

There has been a speculation that pantothenate
synthesized by intestinal microflora may contribute to the
dietary requirement and reduce pantothenate requirement,
therefore reducing the incidence of pantothenate
deficiency. Henderson et a1. (1941) determined in rats
the amount of pantothenic acid ingested and excreted in
the feces and found that the pantothenic acid in feces was
almost independent of the diet. Twenty-one day old rats
consuming a pantothenate deficient basal ration (contained
0.3 - 0.4 ug pantothenate/ kg diet) for six weeks excreted
as much pantothenate per gram of feces as rats receiving
the same diet but supplemented orally with z 20 ug Ca-
pantothenate/day. However, Henderson et al. did not report
on the amount of diet consumed by the rats per day. The
amount of diet consumed is influential to the amount
excreted in the feces. The assumption that most of the
pantothenate excreted in the feces was synthesized by
intestinal bacterial action in cecum was also tested by
Henderson et al. (1942) using three deficient rats. They
found that intestinal content between stomach to cecum
contained 1.5 - 5 ug of pantothenate. A large portion of
fecal pantothenate arise between the cecum and colon
(25 - 40 ug). However, they speculated that the pantothenate
produced may not be available to the rat in an appreciable
amount since deficient rats developed symptoms and even

death, regardless of the amount of pantothenic acid present

23
in intestine and excreted in feces. However, researchers
should be aware of the practice of coprophagia by rats and

its possible influence on the study.

ABSORPTION OF PANTOTHENIC ACID

Pantothenate is present in foods mostly as CoA which
cannot be directly absorbed through mucosal cells. Turner
and Hughes (1962) investigated the absorption of CoA with
everted sacs of rat intestine. They traced the path of
CoA (8 x 10-5 M) which was incubated for an hour in the
mucosal fluid and determined the amount of the coenzyme
remaining on the mucosal side and amount appearing on the
serosal side. Their results, as determined by
microbiological assay using _Lagtgbagillu§_ arabingsus,
showed that 42 - 71% of the CoA remained on the mucosal
side and only 1 - 2% appeared on the serosal side after
an hour. Most of the remaining CoA (8 -31%) appeared in
serosal side as pantothenic acid. The fact that 100% of
CoA was not accounted for in Turner and Houghes' study
(1962) may be due to the low sensitivity of the
microbiological assay. This means that the intestinal
mucosa converts CoA to free pantothenate which is then
passed to the serosal side. It is possible that CoA was
converted to any intermediates such as pantothenine, which
was not assayed for. Intestinal tissues contain a high
level of alkaline phosphatase and pantotheinase which can

hydrolyze CoA to pantetheine or pantothenate (Fenstermacher,

24
1987). In addition, the 1 -2% of CoA found on the serosal
side might have been resynthesized in mucosal cells,
absorbed intact without hydrolysis, or represented the range
of experimental error.

Shibata et a1. (1983) used rats to study the absorption
of pantothenate by using radiolabelled CoA in vivo. They
reported that the absorption occurred by simple diffusion.
They suggested CoA was hydrolyzed in the intestinal lumen
by the following reactions prior to its absorption in the
form of pantothenate:
CoA--->dephospho-CoA--->4’-phosphopantetheine--->
pantetheine--- >pantothenate + cysteamine
Shibata et al. demonstrated that within 2 - 3 hours after
the injection of C-14 labelled CoA into the intestine, 87%
of the isotope appeared in the blood as pantothenic acid.
The values are different from Turner and Hughes’

(1962). This could simply be due to the assays employed,
time of incubation, used of everted sacs versus live animal
and labelled versus non-labelled CoA. There is, however,
no experimental evidence to date, in human, on how CoA is
digested and absorbed.

In an attempt to understand the metabolism and
distribution of pantothenate among organs, Shibata et a1.
(1983) injected 110 nmol of [C-14] pantetheine (0.41 uCi)
in 1.0 m1 of 0.9% NaCl into the duodenal lumen of male

Sprague-Dawley rats weighing 200 - 400 g. Five hours later,

25
they reported that 34.7% C-14 was largely taken up by muscle
and 12.7% by liver.

Hatano (1962) also examined the fate of subcutaneously
injected pantothenic acid in rats previously fed
pantothenate deficient or sufficient diet. Rats in the
control group consumed a diet of 40 mg Ca-pantothenate/kg
diet and the deficient group rats were fed a diet containing
less than 0.8 ug/kg diet. Both groups were maintained on
the respective diet for 32 days starting at 28 days after
birth. Upon loading via subcutaneous injection of 4 mg
Ca-pantothenate into the normal control rats, urinary
elimination of pantothenate over the following 72 hour
period was increased to 2136 ug/72 hours from the normal
of 285 - 354 ug/72 hours, before the load. In the
deficient group, the same dose subcutaneous injection caused
a similar increase, however, it was about 1900 ug/72 hours
or 10% less than that of the control.

The effect of a massive doses (500 mg Ca-pantothenate)
on blood and the distribution between whole blood, plasma
and blood cells was examined in rabbits by Pearson (1941).
Rabbits were examined 60, 75, and 90 minutes after injection
and only 90 minutes after ingestion of 500 mg Ca-
pantothenate. Sixty minutes after injection, whole blood
level of pantothenate increased 114 fold (6400 ug/lOOml)
from control. Coincidentally, the ratio of plasma
pantothenate increased to 5.17 from normal ratio of about

1.0 (range: 0.81 - 1.29). Seventy-five minutes after

26
injection, blood drawn from the rabbits showed a even
greater increase (10,000 ug/100ml) in whole blood. The
plasma/blood cell ratio had increased to 12.00. However,
ninety minutes after ingestion of 500 mg of Ca-pantothenate,
the whole blood pantothenate concentration increased only
10-fold over the normal level of 48 ug/100 ml. Also at
this time, most of the excess pantothenate was located in
the plasma (680 ug/100 ml from normal level of 50 ug/ml).
The maximum rate of excretion of pantothenate in urine
occurred during the first two hours after ingestion of the
massive dose. The level in the urine was maintained fairly
high for approximately 72 hours of collection. Amount
excreted was similar for rabbits injected or ingested 500
mg Ca-pantothenate (296 mg/24 hours, 211 mg/24 hours,

respectively).

SYNTHESIS OF CoA
Lipmann and Kaplan (1948) reported that CoA is
synthesized in rat liver cells from pantothenic acid as
foll ws:
ATP ADP L-cysteine, ATP ADP, Pi

Pantothenate:§b----JZ> 4'-phosphopantothenate- -----Z:->
C02

4’-phosphopantothenylcysteineJa-->4’-phosphopantetheine
ATP 'PPi ATP ADP

-:§- ----- Z> dephospho-CoA ------ ‘Z---> COA

27
The mechanisms regulating CoA synthesis have not been
completely elucidated. CoA synthesis may possibly be
regulated by dietary supply of pantothenate, uptake of the
vitamin by tissue, or combination of nutritional and
hormonal factors such as fasting or diabetes as demonstrated

by Reibel et a1. (1982).

FUNCTIONS OF CoA

CoA is required in many different metabolic reactions
as a transient carrier of acyl groups through a reactive
thiol group (Lehninger, 1982). During acyl-group transfer
reactions, the thiol group is covalently linked to an acyl
group to form thioesters. For example, CoA functions as
an acyl acceptor for pyruvate dehydrogenase complexes and
alpha-ketoglutarate dehydrogenase complexes in the citric
acid cycle. It then forms acetyl CoA and succinyl CoA,
respectively.

Acetyl-CoA is an important intermediary metabolite.
It is formed in the first reaction of oxidative
decarboxylation of pyruvate. Fatty acid can also yield
acetyl-CoA via beta oxidation. Other sources of acetyl-
CoA are the carbon skeleton of ten amino acids (alanine,
threonine, glycine, serine, cysteine, phenylalanine,
tyrosine, leucine, lysine, and tryptophan). These acetyl-
CoA can go through the citric acid cycle where the acetyl
group of acetyl-CoA can then be transferred to oxaloacetate

to yield citrate. The acetyl-CoA can also be used as a

28
carbon source in fatty acid synthesis or incorporated in

cholesterol, steroid hormones, prostagladins synthesis.

TOXICITY OF PANTOTHENIC ACID

Schwartz and Bagdon (1964) examined the acute toxic
effects of pantothenate derivatives. They injected large
intramuscular doses (2.0g/kg and 0.2 g/kg) of sodium
pantothenate, d—panthenol, and d-pantetheine, separately,
into two groups of Sprague-Dawley rats: one group weighed
110 - 120 g, the other weighed 225 - 275 g. The injections
of all derivatives, when examined 96 hours after
administration, did not significantly affect body weight,
liver weight, nor produce evidence of hepatotoxicity.
Hepatotoxicity was determined by histology stained sections
with hematoxylin-eosin or hematoxylin-sudan IV. However
they did notice a slight but not significant increase in
liver lipid deposition in rats weighing 110 - 1209 when
given either 0.2g or 2.0g of d-panthenol per kg body
weight. But this was not observed in heavier rats weighing
220 - 275 g (Schwartz and Bagdon, 1964). Alhadeff et a1.
(1984) reported that, though pantothenate has minimal
toxicity in human, intake of 10 - 20 g per day may lead

to diarrhea and water retention.

MATERIALS AND METHODS

PRELIMINARY EXPERIMENTS

Two preliminary experiments were carried out to
establish valid methodologies. The purpose of the first
experiment was to determine the optimal activities of
enzymes necessary to release all bound forms of pantothenate
to free form in various organs. The purpose of the second
experiment was to determine the extent of conversion of
bound pantothenate to free form in various sample
preparation methods.

Due to different concentrations of bound pantothenate
and endogenous enzymes activities of each organ, we decided
to determine the enzyme activity levels for each organ
sample to maximally release bound pantothenate for the assay
of total pantothenate. To achieve the stated objective,
various organs were excised from normal rats.
Pantetheninase (provided by Dr. Carl Wittwer, Univ. of
Utah), in varying activities (0 to 60 units) was added to
an organ homogenate prepared from approximately 0.5 grams
of wet organ. Simultaneously 0 to 30 units of alkaline
phosphatase (type VII-S, P-5521, Sigma Chemical Co.) were
also added. The samples were incubated (370 C) over night
(10-15 hr), then deproteinized with equimolar of saturated
Ba(OH)2 and 10% ZnSO4 and later analyzed for content of

29

30
pantothenate. We found that a combination of 15 units of
alkaline phosphatase and 30 units of pantetheninase resulted
in the maximal release of pantothenate in all organs.
Additional amount of enzymes to the sample did not increase
the amount of pantothenate released.

Many organs contain alkaline phosphatase and
pantothenase which release pantothenate from its bound
derivatives. It was therefore speculated that post-mortem
autolysis of bound pantothenate from compounds such as CoA
to pantothenate would occur. Many studies have reported
the content of free and bound forms of pantothenate in
various organs, however, the extent of autolysis in various
organs which may have occurred prior to quantification has
not been reported in these studies. The objective of this
preliminary experiment was to determine the extent of
autolysis of bound pantothenate to free pantothenate at
time 0, 1, 2, 6, 12 and 24 hours when organ samples were
held on ice and also after 24 hours, 48 hours, and 2 weeks
when samples were frozen (-4.40 C). Fresh liver was excised
from two 7 week old female Sprague-Dawley rats weighing
an average of 126 g and maintained on a stock diet (Wayne
Lablox) ad libitum. Liver was chosen because it is the
largest organ found in the rat that many samples could be
prepared from. Rats were anesthetized with ether and
sacrificed by bleeding via cardiac puncture. Each fresh
liver was rinsed in saline and divided into equivalent size

(approximately 0.5 gram each) for analysis at different

31
intervals. Liver samples were kept on ice bath or samples
frozen and homogenized at the intervals described earlier
and analyzed for both free and total pantothenate. Results
from this experiment showed that a minimal conversion of
bound pantothenic acid to free pantothenic acid occurred
before 2 weeks of storage at -4.4o C. Therefore, in the
main study, samples were homogenized and prepared within
2 weeks to eliminate possible conversion of bound

pantothenate to free pantothenate.

RESEARCH DESIGN

Forty-eight weanling (21 day old) Sprague-Dawley male
rats (Harlan-Davis, Indianapolis, IN) weighing an average
of 429 were housed in individual hanging stainless steel
cages. Upon receipt, the animals were weighed and
maintained on Wayne Lablox nonpurified stock diet ad libitum
for 24 hours. The rats were maintained on a schedule of
12 hours light, 12 hours dark and at a controlled
temperature (20-220 C). Animals were randomly divided into
four groups (12 per group) to receive for maximum of 8
weeks: 1) group A, AIN-76 purified diet (U.S. BioChemical,
see Table 1, 1a, 1b) with an excessive amount of
pantothenate (+120 mg d-Ca-pantothenate /kg diet), ad
libitum; 2) group B, an adequate control diet (+12 mg d-~
Ca-pantothenate/kg diet), ad libitum: 3) group C, a
pantothenate deficient diet (+0 mg/kg diet), ad libitum;

and 4) group D, an adequate control diet, pair-fed with

32

Table l. Purified pantothenate deficient basal diet
composition (AIN -76TM, U.S. BioChem Co.)

Casein (vitamin free) 20.0
DL-Methionine 0.3
Corn starch 15.0
Sucrose 50.0
Fiber 5.0
Corn oil 5.0
AIN mineral mix* 3.5
AIN vitamin mix**
(without pantothenate) 1.
Choline bitartrate 0.
* see Table 1a
** see Table 1b

Table 1a. AIN-76 TM mineral mixture

Ingredient g/kg
Calcium phosphate, dibasic 500.00
Sodium chloride 74.00
Potassium citrate, monohydrate 220.00
Potassium sulfate 52.00
Magnesium oxide 24.00
Manganous carbonate 3.50
Ferric citrate 6.00
Zinc carbonate 4.60
Cupric carbonate 0.30
Potassium iodate 0.01
Sodium selenite 0.01
Chromium potassium sulfate 0.55
Sucrose to make 1000.00

Table 1b. AIN-76 TM vitamin mixture

Vitamin per kg mixture
Thiamin- HCl 600mg
Riboflavin 600mg
Pyridoxine-HCl 700mg
Nicotinic acid 3000mg
Folic acid 200mg
D-Biotin 20mg
Cyanocobalamin 1mg
Vitamin A 400,00010
Vitamin E 5,000IU
Vitamin D-3 2.5mg
Vitamin K 5mg

Sucrose to make 10009

33
group C. Adequate pantothenate has been recommended by
the National Academy of Sciences-National Research Council
as 8.0 mg Ca-pantothenate/kg diet (National Academy of
Sciences, 1972) while the American Institute of Nutrition
recommended 16 mg Ca-pantothenate/kg diet for laboratory
rats. Thus as a compromise of the two recommendations,
12 mg Ca-pantothenate/kg diet, was used in this study.

Tap water was readily available for all rats.

DATA COLLECTION

Food intake was measured weekly except for the pair-
fed rats and those fed the deficient diet. Intake for each
deficient rat was measured daily so that the same amount
of pantothenate adequate control diet could be fed to its
pair-fed rat the next day. Spillage of food was collected
and accounted for in calculations of food intake. Body
weight was also measured weekly. Clinical observations
such as hair loss, hair color change, behavior changes were
made and recorded by the main investigator daily for the
entire study. However no measurement of these clinical
symptoms was performed.

Five days prior to sacrifice, at week 2, 5 and 8, four
rats/group were housed individually in stainless steel
metabolic cages for collection of urine for two 24 hour
periods. At week two of the experiment, however, only one
48 hour sample was collected from each rat because of the

small volume of collectable urine. An antibacterial agent,

34
1% thimersol (500 ul), was added into each glass collection
vial of urine collected daily. The complete 24 or 48 hour
samples were diluted to 50 ml with deionized water and
stored frozen until analysis.

The same four rats per group were fasted overnight
then anesthetized with ether before sacrifice by bleeding
via cardiac puncture. Blood was collected into vacutainers
containing ethylenediaminetetraacetate (EDTA). Freshly
collected blood was later divided into two portions: one
portion was separated into plasma and packed cells by
centrifugation at 4000 rpm for 10 minutes and the other

left as whole blood.

COLLECTION AND TREATMENT OF ORGANS

Organs (heart, spleen, kidneys) were excised, rinsed
in normal saline, weighed and frozen (-4.4o C) until ready
to be analyzed. Approximately 0.5 grams of wet organ
samples were used for analysis of pantothenate. Since there
were two kidneys from a rat, each kidney (including the
capsule) was weighed and used separately. One entire kidney
was used for determination of free and the other for total
pantothenic acid. This procedure was necessary because
in a preliminary study we found that pantothenate was not
equally distributed within the kidney. We further tested
this by removing the three major parts of the kidney: cortex
(including the capsule), medulla and pelvis, with the aid

of a magnify glass, and analyzed pantothenate content.

35
We discovered that pantothenate concentration differed in
these three major parts of the kidney. As shown in Figure
3, the cortex and pelvis contained a larger concentration
of pantothenic acid than the medulla. Interestingly, the
ratio of bound to free was lower in the cortex compared
to the medulla and pelvis.

Both free and bound pantothenate were measured in
organs and blood using RIA. In urine and plasma only the
free form was estimated because the bound form was not
found. The pre-weighed organs (approximately 0.5 grams)
were later homogenized with distilled water (1000 ul for
all organs) using a Polytron (Brinkmann Instruments Co.,
type PT 1020 3500) at speed 3.5 for one minute for all
organs. For free pantothenate determination, the organ
homogenates were immediately deproteinized with
approximately 0.5 ml each of equimolar amounts of saturated
Ba(OH)2 and 10% ZnSO4. Equimolar ratio was determined by
titrating 10 ml of 10% ZnSO4 diluted with an equal volume
of distilled water against saturated Ba(OH)2 with
phenolphthalein as an indicator. The homogenate was then
centrifuged (Sorvall Superspeed RC2-B Automatic Refrigerated
centrifuge set at 00 - So C) at 4000 rpm for 10 minutes
and the resulting clear supernate was separated and frozen
until used in the radioimmunoassay. For determination of
total pantothenate in organs, the homogenized organ
(approximately 0.5 gram of wet organ) was incubated at 370

C with 15 units of bovine intestine alkaline phosphatase,

36

PA concentration (ug/g tissue) __~

 

 

 

 

 

Cortex Medulla Pelvis
Rat Kidney
- Total PA :3 Bound PA : Free PA

Figure 3. Pantothenate content in cortex, medulla and

pelvis of rat kidney. Total and free pantothenate analyzed
via RIA from 2 normal adult female rats. Bound pantothenate
determined by [tota1]-[free]. Values represent mean i std

dev.

37
type VII-S (P-5521, Sigma Chemical Co.) and 30 units of
pantetheinase (Dr. Carl Wittwer, Univ. of Utah).
Deproteinization of the digested homogenate was in the same
manner as for free pantothenate determination as described
earlier.

Whole blood sample was hemolyzed via three quick
thaw/freeze cycles. For free pantothenate determination,
fifty ul of the hemolyzed blood was immediately
deproteinized with equimolar saturated Ba(OH)2 and 10%
ZnSO4. Blood samples for total pantothenate determination
were subjected to the enzyme treatment of 20 units of
pantetheinase and 10 units of alkaline phosphatase following
hemolysis and incubated overnight for release of the bound
form (amount determined previously in a preliminary study
in our lab). After incubation, the blood samples were
deproteinized as described earlier and analyzed for total
pantothenate. Blood samples for free pantothenate were
deproteinized as described earlier and analyzed via RIA.

Plasma was deproteinized in the same fashion and
analyzed for free pantothenate only. Plasma from pair-fed
control rats at week 8 of the study was not analyzed due
to collection of an inadequate amount of whole blood. Only
the whole blood was used for analysis. Urine samples,
diluted with distilled water, were analyzed for free
pantothenate by RIA. All supernatants and samples were

completely thawed before analysis by RIA.

3 8

PANTOTHENIC ACID DETERMINATION

Pantothenate was determined by radioimmunoassay (RIA)
as described by Wyse et al.(l979). Fifty ul of supernat
(of organ, whole blood, plasma or urine) was pipetted into
a vial (5 m1 polypropylene, Denville Scientific, Inc.,
#V9951). To each vial, two hundred-fifty ul of 1.2% rabbit
serum albumin in phosphate buffered saline (PBS) solution
mixed with antisera. Rabbit antisera were added to the
vial with ranges from 1:100 - 1:35 dilution to obtain the
desired binding of 30-50% in the blank. Approximately 5000
dpm of labelled 14-C pantothenate (Sodium [1-C14]-D-
pantothenate, specific activity of 3.7 Ci/mole, from New
England Nuclear, Boston, MA) was also pipetted to each
mixture. The final mixture was agitated and incubated for
15 minutes at 370 C. To each vial, three hundred-twenty
ul (a volume equivalent to the sum of supernat, rabbit serum
albumin in PBS, antisera and radiolabelled pantothenate)
of saturated ammonium sulfate were added to result in a
50% saturated ammonium sulfate in the final medium. After
thorough mixing by vortexing each vial, the vials were
centrifuged (Sorvall Superspeed RC2-B Automatic Refrigerated
Centrifuge set at 00 - So C) at 68,000 x g (or 85,000 rpm)
for 15 minutes. The supernatant was then suctioned off
leaving the precipitate pellet and, again, 500 ul of 50%
saturated ammonium sulfate was added to the precipitate
pellet, resuspended and centrifuged at 68,000 x g for 15

minutes. Supernatant was again suctioned off. Tissue

39
solubilizer (soluene 350, Packard Instrument Co, Inc.,
#6003038) was then added (30 ul) to the pellet remaining
in the sample vial. Vials were allow to sit in 600 C water
bath to for 30 minutes to enhance dissolving of the pellet.
Scintillation cocktail (Safety-Solve, Research Product
International Corp., #111177), in the amount of 3.5 ml,
was added to the vials for counting in liquid
scintillation. Radioactivity of the samples was read by
the Packard 4000 series liquid scintillation counter
(Packard Instrument, Inc.) for five minute per vial.
Standards used for assays ranged from 0 to 350 ng d-Ca-
Pantothenate. The standard curve was constructed by
plotting the percent bound as a function of ng pantothenic

acid on log-probit paper.

STATISTICAL ANALYSIS

Statistical analyses of the data were performed using
two-way analysis of variance (ANOVA) to test for treatment
effect, time effect, and the interaction effect between
the ad lib control and the deficient group, and the pair-
fed control and the deficient group. The variables examined
were diet intake, growth, organ weight, organ pantothenate
concentration (total and free), whole blood (total and
free), plasma and urine (free only) pantothenate
concentration. The difference between the treatments was
further tested by Dunnett t-test selecting the deficient

group as the control group to be compared to the ad lib

40
control and the pair-fed control. Correlation within the
deficient group of organ pantothenate concentration with
bodily fluid was calculated using product-moment
correlation. The predictive equation for each organ was
estimated using bodily fluid variables whose simple
correlation with each organ was greater than "0.3". The
predictive equation for bodily fluid was estimated using
variables which displayed the highest simple correlations

with bodily fluid.

RESULTS

Rats fed the diet deficient in pantothenic acid (group
C) exhibited pantothenate deficiency signs similar to those
reported by others (Hatano, 1962; Henderson et al., 1941:
Reibel et a1., 1982). The deficient rats in this study
demonstrated anorexia and consumed less (in grams per day)
than the ad lib control (group B) or the group fed 120 mg
pantothenate per kg diet (group A). The deficient rats
also failed to grow as well as their counterparts fed a
pantothenate supplemented diet ad lib. In addition, the
deficient group rats also experienced occasional nasal
discharge of blood-like fluid. Because albino Sprague-
Dawley rats were used in this study, graying of the hair
was not detected. However a rusty tinge was noted in the
deficient rats by the 3rd week of the study and they lost
more hair. The other groups exhibited no overt
abnormalities.

There was a significant time and diet effect (p<0.01)
on daily intake of the rats in the study. The average daily
intake of the deficient group was significantly (p<0.01)
lower than ad lib control as early as week 2 of the study.
The rats of deficient group consumed a range of 7 to 11'
grams of diet per day over the eight weeks of the study,

whereas rats of ad lib control consumed 11 to 22
41

42
grams of diet per day as shown on Figure 4.

Growth, measured by weight gain, also showed time,
diet as well as the interaction effect. Weight gain of
the deficient rats, were significantly lower (p<0.05) than
the ad lib control rats at week 4 to week 8 of the study
as shown in Figure 5 (group B weight: 197:75: group C
weight: 122:34). Over all, the pair-fed control (group
D) rats gained more weight than the rats of the deficient
group which consumed the same amount of diet but absent
of pantothenate. However the weight difference between
the deficient group and pair-fed control was not
statistically significant using the Dunnett t-test.

The rats fed a pantothenate deficient diet had lower
organ weight in the heart, spleen and kidney than the ad
lib control as shown on Table 2. Significant difference
(p<0.05) in heart weight between the deficient and the ad
lib was observed at weeks 2 and 8 but not 5. Spleen weight
was significantly different (p<0.01) between the deficient
and the ad lib control at all time points. Difference in
kidney organ weight was significant (p<0.10) near the end
of the study (weeks 5 and 8) between the deficient and the
ad lib control. However, there were no significant
differences in organs weights when presented as percent
body weight among the different groups of the organs
examined.

Overall, the rats in group fed 120 mg Ca-pantothenate

per kg diet (+120 mg/kg diet) grew comparable to ad lib

43

Average Amount of Diet Consumed (gram)

 

:2‘, _ — "l ‘ I \‘ - \
‘ c —'—~—~.
o

15*

 

 

wm sz Wk3 Wk4 m5 wxe Wk7 Wk8
Weeks of Study

+GpA *GpB “—‘GpCprDl

Figure 4. Diet intake of rats.

Group A (Gp A) rats were fed 120 mg Ca-pantothenate/kg diet
ad lib, group B (Gp B) rats were fed the ad lib control
diet, ad lib, and group C (Gp C) rats were fed the
pantothenate deficient diet. Same amount consumed by group
C was pair-fed to rats of group D (pair-fed control) with
the control diet. Values represent mean 1 SEM. Statist1cal
significance between Gp B and C: * = p<0.001.

44

Average Body Weight of Rats (grams)

[

 

/’/
250— , ”M”
-/"/7/
—’ ’I -1.
_ ,9/ ,7 .\ ‘:'~~~~ -1 '
200 ;— I ..v/ r:

i .- ~ ' V” °\---- ..
. ,. '1 __.‘_‘_ ;/ F- #‘h‘xw

 

" " . c-o ..-

 

0 _ -. --...-
WkO wm Wk2 Wk3 Wk4 Wk5 Wk6 wn Wk8
Weeks of Study

 

+GpA *GpB +606 "'3—GpD

Figure 5. Growth pattern of rats.

Weight gain of group A (Gp A) rats fed 120 mg Ca-
pantothenate/kg diet, ad lib, group B (Gp B) rats fed the
ad lib control diet, ad lib, group C (Gp C) rats fed the
pantothenate deficient diet, ad lib, and group D (Gp D)
pair-fed the control diet. Values represent mean 1 SEM.
Statistical significance between Gp B and C: o = p<0.10,
* = p<0.05.

45

Table 2. Total weight and percent of body weight of rats
in the study.

Group Al Group B Group C Group D
HEART: gram weight (percent body weight)
Wk 2
0.53:0.20 0.70:0.17* 0.41:0.11 0.41i0.09
(.005:.0006) (.006:.002) (.004:.0005) (.005:.0004)
Wk 5
0.87 0.76 0.55 0.51
(.004) (.004) (.005) (.004)
Wk 8
0.98 0.98* 0.64 0.59
(.004) (.004) (.005) (.004)
SPLEEN
Wk 2
0.44:0.08 0.46i0.10** 0.27:0.05 0.33i0.07
(.0041-0009) (.004:.0007) (.003;.0004) (.004i-0007)
Wk 5
0.58 0.50** 0.28 0.30
(.003) (.003) (.002) (.002)
Wk 8
0.55 0.57** 0.31i0.07 0.37
(.002) (.002) (.003) (.003)
KIDNEY
Wk 2
1.07:0.36 1.15:0.33 0.97:0.19 0.79:0.24
(.01:.001) (.01:.001) (.01:.0009) (.008:.001)
Wk 5
1.68 1.520 1.05 1.03
(.008) (.008) (.009) (.008)
Wk 8
1.88 1.87* 1.20 1.22
(.008) (.007) (.01) (.008)

Values represents mean i SEM

1Group A= rats fed 120 mg Ca-pantothenate/kg diet, ad lib.
Group B= rats fed control, ad lib.

Group C= rats fed deficient diet, ad lib.

Group D= pair-fed the control diet.

Statistical significance between Gp B and C:

o = p<0.10, * = p<0.05, ** = p<0.01.

46

control (+12 mg/kg diet) throughout the 8 weeks. No toxic
signs or symptoms of feeding 10 times the recommended amount
of pantothenate for rats were observed. The excess amount
of pantothenic acid in the diet of group A did not result
in a higher pantothenate concentration in any of the organs
examined compared to the rats fed the control diet ad lib
fed and pair-fed. The pantothenic level in whole blood,
plasma and urine of the group fed 120 mg Ca-pantothenate
per kg diet, however, was significantly greater (p<0.01)
than the ad lib control, suggesting that excess pantothenic
acid was absorbed and excreted. The pair-fed control group
did not differ in the organs examined nor in blood and urine
pantothenate concentration of total and free pantothenic
acid from the ad lib control. Although the pair-fed rats
had lower body weights than the rats fed ad lib control,
the pair—fed control still maintained organ pantothenic
acid concentration comparable to the ad lib control group.

Of the organs examined, the largest concentration of
total pantothenic acid was found in the order of kidney,
heart and spleen. The free to total ratio of pantothenic
acid was similar in groups fed 120 mg Ca-pantothenate per
kg diet, ad lib control, and the pair-fed control as well
as the deficient group in all organs examined (Table 3).
Within each group, no consistent changes were noted with
increase age of the rat.

At a confidence interval of 90%, the total pantothenic

acid concentration (ug of pantothenate/g organ) in the heart

Table 3.

47

The ratio of free (F) to total (T) pantothenate

in heart, spleen and kidney of rats in the study.*

no
M

We.
a
U1

we.
a
m

arenas'urauisvnranas
H

x
M

We.
a
U1

We.
a
m

'flr351€'flr311€'flrifil€
H

w
N

We.
a
U"

F3"fl§jfl*3§l€'fitifil€
00

Heart, ug/g

12.90i5.48 17.83i2.02

39.64i7.00
0.32i0.11

19.99
43.19
0.47

17.85
52.40
0.34

7.61:2.37
16.78i4.26
0.47:0.14

5.98
15.98
0.39

4.02
12.08
0.35

47.25:8.59
0.40:0.07

16.32
51.84
0.32

17.61
56.68
0.31

Spleen

9.28:3.88
16.09i5.36
0.65:0.28

1.68
15.44
0.13

3.24
15.74
0.25

Kidney

47.81:10.56 33.67i7.44
72.15:11.37 54.98i4.68

0.68:0.15

38.04
49.75
0.76

49.06
64.34
0.72

0.62:0.15

48.52
56.86
0.86

44.64
53.19
0.85

organ

10.16:3.15
32.69:8.06
0.34:0.12

12.03
37.49
0.32

11.02
39.68
0.30

0.40i0.50
8.58:2.74
0.05:0.15

1.19
6.89
0.23

1.17
4.27
0.28

21.36i5.57
39.78i13.90
0.55:0.14

30.73
66.78
0.47

30.58
42.03
0.73

15.96i3.03
44.14i7.99
0.37:0.09

14.76
50.29
0.29

17.44
57.53
0.32

11.37i4.39
18.75i5.81
0.61:0.27

3.12
11.97
0.26

4.12
7.40
0.65

35.50i4.97
49.01il3.18
0.75:0.10

37.80
67.37
0.58

30.07
52.92
0.58

represent meaniSEM
A= rats fed 120 mg Ca-Pantothenate/kg diet, ad lib.

B= rats fed control diet, ad lib.
C= rats fed deficient diet, ad lib.

D= pair-fed the control diet.

48
showed only a diet effect. Result of the interaction
contrast shows that there appears to be a slight ordinal
interaction. The total pantothenate concentration was lower
in the deficient group than in the pair-fed control and
ad lib control (Figure 6). The total concentration in the
heart was significant (p<0.05) at weeks 2, 5 and 8 between
the ad lib control and deficient. Significance in heart
total pantothenate concentration was seen later at weeks
5 and 8 between the deficient and the pair-fed group
(p<0.10). At the same confidence interval of 90%, the free
pantothenate concentration also only showed a diet effect.
However, interaction contrast shows almost no effect
(interaction contrast = ~0.34) between deficient and pair-
fed as well as deficient and ad lib control. The
concentration of the free form of pantothenic acid in the
heart of the deficient group was significantly different
(p<0.05) from pair-fed control and ad lib control at weeks
2 and 8 but not 5, similar to the trend seen in the
difference of heart weight.

Content of pantothenate in the whole organ was
determined by multiplying the concentration (ug of
pantothenate per gram of organ) times the gram weight of
the whole organ (Table 4). The heart pantothenate content
of rats fed the pantothenate deficient diet did increase'
(12 ug of pantothenate) throughout the 8 weeks of the
study. The pair-fed control group had an increase of 16

ug of pantothenate, that is 4 mg more than the deficient

40

PA concentration (ug PA/g organ)

   

50 L
40 »
30 ..

20*

 

Weeks of Study

- 0;: A, total 1.4.41 Gp A. free l__.l ep 8. total ep 8, free

ep 0.102.: iii: ep C.free 3 ep D.totsl IEEE 0p D.1rce

   

Figure 6. Concentration of pantothenate in rat heart.
Total and free pantothenate concentration of group A (Gp

A) rats fed 120 mg Ca-pantothenate/kg diet, ad lib, group

B (Gp B) rats fed the ad lib control, ad lib, group C (Gp
C) rats fed the pantothenate deficient diet, ad lib, and
group D (Gp D) pair-fed the control diet. Values represent
mean i SEM. Statistical significance between Gp B and C:

o p<0.10,

* p<0.05. Statistical significance between Gp D and C:

A p<0.10, # = p<0.05.

50

Table 4. Pantothenate content in whole rat heart.

Week 2: Total (Free), ug/organ

21.21i13.03 31.81:ll.l9 l3.48i7.63 18.46i8.45
(6.89:6.25) (12.2717-90) (4.19:1.95) (6.66:1.97)
Week 5
37.43 39.43 20.26 25.36
(17.47) (12.28) (6.50) (7.42)
Week 8
51.29 55.26 25.81 34.26
(17.27) (17.312) (6.97) (10.11)

Values represent meaniSEM.

*Group A= rats fed 120 mg Ca-pantothenate/kg diet, ad lib.
Group B= rats fed control diet, ad lib.

Group C= rats fed deficient diet, ad lib.

Group D= pair-fed the control diet.

51
group, in total heart content. However, the content of
pantothenate in the heart of the rats in ad lib control
increased 23 ug in the eight weeks of the study. The rats
in the group fed 120 mg pantothenate per kg diet and the
pair-fed control had heart pantothenate concentration and
content similar to the ad lib control group. Thus, an
excess supplementation of pantothenate in the diet (as was
for the group fed 120 mg pantothenate per kg diet rats)
did not increase pantothenate concentration nor content
in the heart.

In the spleen, at the confidence interval of 90%, diet
also seems to have an effect. The interaction contrast
shows a strong ordinal interaction between the deficient
and the two control diet fed groups (ad lib and pair-fed).
The deficient group had significantly lower total
pantothenate concentration than ad lib control (p<0.10)
at weeks 2, 5 and 8 of the study (Figure 7). Free
pantothenate concentration had significant (p<0.10) diet,
time and the interaction of the two effect between the
deficient and the two groups fed the control diet.
Interaction contrast shows an association between the two
groups. Upon graphing, it appeared to be a disordinal
response. Significance was seen only at week 2 between
the deficient group and the two control diet fed groups
(p<0.01). Pantothenate content in whole spleen was lower

in the deficient group than pair-fed control, ad lib

 

 

 

 

 

 

 

 

 

Weeks of Study

- Op A. total :3 Op A. tree : Op 8. total Op 8. tree
Op 0. total : Op C. tree 3 Op 0. total 2: Op D. tree

 

Figure 7. Concentration of pantothenate in rat spleen.
Total and free pantothenate concentration of group A (Gp
A) rats fed 120 mg Ca-pantothenate/kg diet, ad lib, group
B (Gp B) rats fed the ad lib control, ad lib, group C (Gp
C) rats fed the pantothenate deficient diet, ad lib, and
group D (Gp D) pair-fed the control diet. Values represent
mean i SEM. n=4/group. Statistical significance between
Gp B and C:

o = p<0.10, * = p<0.05, ** = p<0.01. Statistical
significance between Gp D and C: A = p<0.10, # = p<0.05,
## = p<0.01.

53

control, as well as rat fed 120 mg pantothenate per kg diet
in all weeks examined as shown in Table 5.

The total pantothenate concentration in kidney showed
a time effect at the confidence interval of 90%.
Interaction contrast shows almost zero effect. The
deficient group, the pair-fed control and ad lib control
was not significantly different at any time in kidney
pantothenate concentration. Free pantothenate
concentration, between the deficient and the ad lib control,
had a time and diet effect at the confidence interval of
90%. Interaction contrast shows a strong ordinal
interaction. The deficient group was significantly lower
(p<0.05) than ad lib control at weeks 2, 5 and 8. However,
this significance was not observed between the deficient
and the pair-fed control (Figure 8). Content of pantothenic
acid in whole kidney increased 8 ug (from 38 ug to 46 ug)
while the organ weight increased 0.23 gram in the deficient
group. Content of pantothenate in the kidney of the rats
of the pair-fed control increased 25 ug which is 3 times
the increased seen in the deficient group. Weight of the
kidney in the pair-fed control rats increased 0.43 grams.
When the rats are allowed to consume as much of the control
diet as desired, as with the rats of the ad lib control,
pantothenate content in the kidney increased 37 ug (from
62 ug to 99 ug) which is close to 5 times the increase seen
in the deficient group. In addition, the organ weight of

the ad lib control increased 0.72 grams which is 3 times

54

Table 5. Pantothenate content in whole rat spleen.

Week 2: Total (Free), ug/organ

7.27:2.20 7.27:2.47 2.36:0.87 6.17i1.81
(3.30:1.07) (4.18:1.65) (0.11:0.15) (3.73:1.45)
Week 5
9.00 5.65 1.98 3.54
(3.44) (0.76) (0.33) (0.92)
Week 8
6.69 8.64 1.37 2.67
(1.92) (1.93) (0.37) (1.53)

Values represent mean:SEM.

*Group A= rats fed 120 mg Ca-pantothenate/kg diet, ad lib.
Group B= rats fed control diet, ad lib.

Group C= rats fed deficient diet, ad lib.

Group D: pair-fed the control diet.

 

 

Weeks of Study

- ep A. total l...) (39 A. tree ': Gp a. total Op B. mo
99 c. tom Z: Gp c. m. E up 0, mm m (in 0. mo

 

Figure 8. Concentration of pantothenate in rat kidney.
Total and free pantothenate concentration of group A (Gp

A) rats fed 120 mg Ca-pantothenate/kg diet, ad lib, group

B (Gp B) rats fed the ad lib control, ad lib, group C (Gp
C) rats fed the pantothenate deficient diet, ad lib, and
group D (Gp D) pair-fed the control diet. Values represent
mean i SEM. n=4/group. Statistical significance between

Gp B and C:

* = p<0.05, ** = p<0.01. Statistical significance between
Gp D and C: # = p<0.05.

56
the weight increase seen in the deficient rats (Table 6).
Without pantothenate in the diet, the growth of the kidney
appeared hindered.

The whole blood total pantothenate concentration
showed a diet effect between the deficient and the ad lib
as well as between the pair-fed at the confidence interval
of 90%. The deficient group was significantly lower
(p<0.05) than ad lib control in all weeks examined (Figure
9). Significant difference in total pantothenate
concentration (p<0.01) between the deficient group and the
pair-fed control was found only at week 2. At the same
confidence interval of 90%, the free pantothenate
concentration had a diet effect. Free pantothenate
concentration of whole blood in the deficient group was
not significantly lower compared to the pair-fed control
nor the ad lib control at any time point. The group fed
120 mg Ca-pantothenate per kg diet had the largest
pantothenate concentration in whole blood, followed by ad
lib control, pair-fed control and finally the deficient.
The ratio of free to total pantothenate in whole blood was
about 1 in all the groups examined.

At the confidence interval of 90%, the plasma
pantothenate concentration had a diet and time effect
between the deficient and the two control diet fed groups.
Plasma free pantothenate concentration was significantly
lower (p<0.05) in the deficient group compared to the ad

lib control at weeks 5 and 8 of the study as shown on Figure

57

Table 6. Pantothenate content in whole rat kidney.

Week 2: Total (Free), ug/organ
76.91i23.03 62.35:l7.42 38.46:l4.26 40.70i20.21

(50.39:22.27)(38.53:21.33) (20.78i7.78) (28.84:8.75)

Week 5
83.82 86.67 69.69 69.33
(64.00) (74.01) (32.28) (38.72)
Week 8
120.77 99.34 46.34 65.62
(92.53) (83.87) (35.95) (37.18)

Values represent mean:SEM.

*Group A= rats fed 120 mg Ca-pantothenate/kg diet, ad lib.
Group B: rats fed control diet, ad lib.

Group C= rats fed deficient diet, ad lib.

Group D= pair-fed the control diet.

PA concentration (ug PA/ml whole blood)

1 ___.____ w. 11___ 11, __

t

1.4?

 

 

 

 

 

 

1.2 r n
t
1 )—
08% ; -
06? i F
0.4 f— \t ::::::
(x: ::::: v - ..... 1
\ ::::: I) ’4
0.2 .. ( .gm; (3
0: \ E? “3 .: 5%
Wk 2 Wk 5
Weeks of Study
- op A. total ZZZ) am A, m. : 8p 8. total 8p 8. tree
.... 8p 0. total : Op 0. tree fl Op D. total :31 Go 0. "00

Figure 9. Concentration of pantothenate in rat whole blood.
Total and free pantothenate concentration of group A (Gp

A) rats fed 120 mg Ca-pantothenate/kg diet, ad lib, group

B (Gp B) rats fed the ad lib control, ad lib, group C (Gp
C) rats fed the pantothenate deficient diet, ad lib, and
group D (Gp D) pair-fed the control diet. Values represent
mean i SEM. n=4/group. Statistical significance between

Gp B and C:

* p<0.05. Statistical significance between Gp D and C:

# p<0.05.

59
10. No plasma was collected at week 8 of the pair-fed
group, thus comparison could not be made. The rats of group
fed 120 mg pantothenate per kg and the ad lib control
contained the highest plasma pantothenate concentration,
followed by the pair-fed control and lastly the deficient
group in all weeks examined.

Urine pantothenate excretion was significantly lower
(p<0.01) in the deficient group than the pair-fed and ad
lib control rats at weeks 5 and 8 (Figure 11). The group
fed 120 mg pantothenate per kg diet, consistently, had
significantly higher (p<0.01) concentration (ug
pantothenate/24 hours) of free pantothenate excretion (334 -
375 ug/24 hours) than the ad lib control (range 18 - 121
ug/24 hours). The ad lib control and the pair-fed control
had comparable amount of free pantothenate excreted at week
2 and 5. However, by week 8 of the study the effect of
limited intake on the pair-fed rats was apparent (the pair-
fed excreted less than the ad lib control). By week 8,
the pair-fed group was fed approximately 7 grams of diet
per day (compared to about 20 grams consumed by the ad lib
control rats). It appeared that the pair-fed rats were
conserving pantothenate stores by a decreasing urinary
pantothenate excretion when the amount of diet was
restricted. The deficient group had significantly lower
(p<0.01) pantothenate excreted than either control group.
This was noted at week 2 (4.05 i 1 ug/24 hours compared

to pair-fed control: 21.6 i 23 ug/24 hours and ad lib

60

1 PA concentration (up PA/ml plasma)

 

 

 

 

Weeks of Study

-Op A. tree 2 Op 8. fro. : Op c. troo -Op 0. tree

Figure 10. Concentration of pantothenate in rat plasma.
Free pantothenate concentration of group A (Gp A) rats fed
120 mg Ca-pantothenate/kg diet, ad lib, group B (Gp B) rats
fed the ad lib control, ad lib, group C (Gp C) rats fed

the pantothenate deficient diet, ad lib, and group D (Gp

D) pair-fed the control diet.- n=4/group. Values represent
mean 1 SEM. Statistical significance between Gp B and C:

* = p<0.05,

** = p<0.01.

61

Urinary excretion of PA (up PA/24 hrs)
400 T
t

300 f‘
200?

100}-

 

/}

 

\W__ . (
Wk 2 Wk 5
Weeks of Study

//, V, .

 

-Op A, m. -_._.;.;J Op 8. tree : Op C. troa Op 0. tree

Figure 11. Concentration of pantothenate in rat urine.
Free pantothenate excreted in urine per 24 hours of group
A (Gp A) rats fed 120 mg Ca-pantothenate/kg diet, ad lib,
group B (Gp B) rats fed the ad lib control, ad lib, group
C (Gp C) rats fed the pantothenate deficient diet, ad lib,
and group D (Gp D) pair-fed the control diet. n=4/group.
Values represent mean i SEM. n=4/group. Statistical
significance between Gp B and C: * = p<0.05, ** = p<0.01.
Statistical significance between Gp D and C: # = p<0.05.

62
control: 19.8 i 37 ug/24 hours). The low level of urinary
pantothenate remained constant in the deficient group for
the duration of the study. However, rats consuming adequate
pantothenic acid (ad lib control and pair-fed control)
increased the concentration of pantothenate excreted over
the weeks of the study. During period of fast growth, at
5 weeks of age or at week 2 of the study, more pantothenate
may be needed for organ growth and increased metabolism
than during a period of less growth. Thus, due to
conservation of pantothenate, less pantothenate was excreted
at week 2 in the ad lib and pair-fed control than the
following weeks.

Correlation of the pantothenate concentration of whole
blood, plasma and urine to organs concentration was
important in this study to determine if one could predict
the other. It was also important to note if easily
accessible bodily fluids were good indicators of
pantothenate status. Pantothenate values in whole blood,
plasma and urine were correlated to organ pantothenate
concentration. The deficient group was the population at
risk for pantothenate deficiency and was focused on. Levels
of pantothenic acid in organs, blood and urine may have
reached saturation and plateaued in the rats of the groups
fed the pantothenate supplemented diet. Thus, normal rat
values of pantothenate concentration were not considered
for determining the best pantothenate indicator and

predictor. The results of the correlations are shown on

63
Table 7. Under the experimental conditions, the highest
correlation was seen between plasma pantothenate
concentration and free pantothenic acid concentration in
spleen (r = 0.80). Total pantothenate concentration in
whole blood correlated best with free pantothenate
concentration in kidney (r = 0.48). Free pantothenate
concentration of whole blood correlated best with heart
(r = 0.60). Urine was poorly correlated with organ
pantothenate concentration and was more reflective of the
diet and this supports Eissenstat et al.’s (1986) study
with human subjects.

Since the variables of interest (pantothenate
concentration in organs) were determined this study, the
concern of the equation formulated is the ability (of plasma
pantothenate concentration) to predict future random
values. The variables with the highest correlation were
used in determining the predictive equations. In predictive
equation 1, actual free pantothenate concentrations of heart
(x1), spleen (x2) and kidney (x3) were inserted into the
equation from the independent observations made from the
rats of the deficient group (n=12). Organ free pantothenate
concentration (x1, x2, x3) was paired to plasma pantothenate
concentration (y). These pairs were then used to estimate
partial regression coefficients. We assumed a linear
function existed between the pairs so that the data points
were fitted into a hyper-plane where Y1 = b0 + b1[xl] +

b2[x2] + b3[x3]. The estimates of parameters determined

64

Table 7. Correlation results of organ pantothenate (PA)
concentration vs bodily fluid of group C rats (r).

Whole Blood Free PA
Total PA Free PA Plasma Urine

Total PA

Heart -0.06 -0.18 0.22 0.04
Spleen -0.40 -0.12 -0.49 0.25
Kidney 0.01 0.52 0.49 -0.42
Free PA

Heart 0.37 0.60 0.78 -0.41
Spleen 0.44 0.56 0.80 0.12

Kidney 0.48 0.37 0.68 —0.04

65
should produce a sum of squares that is minimized by the
method of least square. Predictive equation 1 formulated
to predict pantothenate status of the rat is as follows:
(Free pantothenate in plasma) =
' b0 + [b1(free pantothenate in heart)

+ b2(free pantothenate in spleen)
+ b3(free pantothenate in kidney)]

where,

b0 = -0.0183
bl = 0.00216
b2 = 0.0186
b3 = 0.000299

Table 8 shows the residual results of the predictive
equation 1. The coeffcient of determination (R2) of
predictive equation 1 is 0.80. This means that 80% of the
variation in Y is explained by the variation in x1, x2 and
x3.

Prediction equations 2, 3 and 4 are the
reverse of prediction equation 1. In 2, 3 and 4 we
determined the ability to predict organ pantothenate
concentration when the concentration of pantothenate in
bodily fluids (whole blood, plasma, and/or urine) is known.
These equations will be more realistic in a clinical setting
because rarely is a biopsy performed to determine
nutritional status of the individual. Again, only highly
correlated variables were used (r > 0.30) to determined
predictive equations 2, 3 and 4.

Predictive equation 2 uses the free pantothenate
concentration found in whole blood, plasma, and urine to

calculate the free pantothenate concentration in the heart

66

Table 8. Residual results of the predictive equation 1*.
Actual Y Predicted Y Residual Value
Value (ug/ml) (ug/m1)
0.017 0.010 0.007
0.010 0.015 -0.005
0.015 0.025 -0.010
0.004 -0.000 0.004
0.029 0.029 -0.000
0.033 0.025 0.008
0.040 0.031 0.009
0.074 0.071 0.003
0.023 0.029 -0.006
0.018 0.028 -0.010
0.029 0.044 -0.015
0.059 0.045 0.014
*Predictive equation 1: Y1 = b0 +[b1(x1)+b2(x2)+b3(x3)]
where: Y1 = Free PA in plasma (ug/ml)

x1 = Free PA in heart (ug/g)

x2 = Free PA in spleen (ug/g)

x3 = Free PA in kidney (ug/g)
and

b0 = -0.0183

bl = 0.0022

b2 = 0.0186

D3 = 0.0003

67
of a deficient rat. The predictive equation 2 is as
follows:

(Free PA in heart) =
b0 + [b4(free PA in whole blood)
+ b5(free PA in plasma)
+ b6(free PA in urine)]

where,

b0 = 6.73
b4 = 19.73
b5 = 78.85
b6 = -0.74

The residual results are shown on Table 9. The R2 for
predictive equation 2 is 0.63.

Predictive equation 3 estimates the free pantothenate
concentration in the spleen of a deficient rat from total
and free pantothenate concentration in whole blood as well
as free pantothenate concentration in plasma. Urine was
not highly correlated with spleen (r = 0.12) and thus, not
used in the equation. The residual results are shown on
Table 10 and the R2 for this equation is 0.65. Predictive
equation 3 is as follows:

(Free PA in Spleen) =
b0 +[b7(total PA in whole blood)

+ b8(free PA in whole blood)
+ b9(free PA in plasma)]

Where,

b0 = 0.097
b7 = -0.013
b8 = 0.122
b9 = 19.263

The total and free pantothenate concentration in whole
blood along with the free pantothenate concentration in

plasma were used to formulate the predictive equation (4)

68

Table 9. Residual results of the predictive equation 2*.

Actual Y Predicted Y Residual Value
Value (ug/g) (ug/q)
6.73 9.17 -2.44
9.33 8.87 0.46
14.45 10.15 4.30
10.13 9.24 0.89
8.19 9.54 -1.35
10.23 12.80 -2.57
10.96 13.27 -2.31
18.74 17.19 1.55
9.94 10.81 -0.87
8.80 8.05 0.74
10.97 10.42 0.55
14.37 13.34 1.03

*Predictive equation 2: Y2 = b0 +[b4(x4)+b5(x5)+b6(x6)]
where: Y2 Free PA in heart (ug/g)

x4 = Free PA in whole blood (ug/ml)

x5 = Free PA in plasma (ug/ml)

x6 = Free PA in urine (ug/24 hr)
and

b0 = 6.73

b4 = 19.72

b5 = 78.85

b6 = -0.74

69

Table 10. Residual results of predictive equation 3*.

Actual Y Predicted Y Residual Value

Value (ug/g) (ug/q)
0 43 0.68 -0.25
0 34 0.48 -0.14
0.31 0.64 -0.33
0.51 0.48 0.03
1.14 0.93 0.21
0.70 0.99 -0.29
0.93 1.16 0 23
1.99 1.90 0.09
0.86 0.79 0.07
0.92 0.61 0 31
1 65 0.91 0.74
1.25 1.45 -0.20

* Predictive equation 3: Y3 = b0 +[b7(x7)+b8(x8)+b9(x9)]
where: Y3 Free PA in spleen (ug/g)

x7 = Total PA in whole blood (ug/ml)
x8 = Free PA in whole blood (ug/ml)
x9 = Free PA in plasma (ug/ml)
and
b0 = 0.097
b7 = -0.013
b8 = 0.122
b9 = 19.263

70

for free pantothenate concentration in the kidney of the
deficient rat. The residual results are shown on Table
11. The R2 (= 0.48) of predictive equation 4 is not as
strong as the earlier equations but it is worthwhile to
note. Predictive equation 4 is as follows:

(Free PA in Kidney) =

b0 + [b10(total PA in whole blood)

+ b11(free PA in whole blood)
+ b12(free PA in plasma)]

Where,

b0 = 21.63
bll = 0.63
b12 = -0.76
b13 = 183.46

71

Table 11. Residual results of predictive equation 4*.

Actual Y Predicted Y Residual Value

Value (ug/g) (ug/g)
20.56 24.92 -4.36
22 86 24.00 -1.14
20.78 24.59 -3 81
21.24 22.16 -0.92
29.13 27.76 1.37
26.26 28.23 -1 97
28.61 28.64 -0.03
38.91 35.41 3 50
34 14 27.38 6.76
33.34 25.53 7.81
25.86 27.31 -1.45
28.99 34.76 -5.77

*Predictive equation 4:
Y4 = b0 + [b10(x10)+b11(x11)+b12(x12)]
where: Y4 Free PA in kidney (ug/g)

x10 = Total PA in whole blood (ug/ml)
x11 = Free PA in whole blood (ug/ml)
x12 = Free PA in plasma (ug/ml)
and
b0 = 21.63

b7 = 0.63

b8 = -0.76

b9 =183.46

DISCUSSION

It is evident from this study that pantothenic acid
is an essential nutrient for male Sprague-Dawley rats. The
Ca-pantothenate supplementation of 12 mg/kg diet was
sufficient to prevent deficiency signs as observed in rats
of group C, fed the pantothenate deficient diet.
Supplementation of 120 mg Ca-pantothenate/kg diet (group
A) in rats showed no deviation from the rats supplemented
with 12 mg Ca-pantothenate/kg diet (group B). Similar
results were seen by Barboriak et al. (1956). They reported
that weanling rats fed 100 mg Ca-pantothenate/kg diet ad
libitum also failed to gain as much weight, also not
significantly, as their counterparts fed 8 mg Ca-
pantothenate/kg diet ad libitum. Thus, Ca-pantothenate
greater than 8 mg/kg in the diet did not result in greater
growth or weight gain for the 680 days of their study.
In fact, the reverse was seen in this study as well as in
the study of Barboriak et al. Barboriak et al. also fed
weanling rats Ca-pantothenate levels of 2 mg/kg and 4 mg/kg
ad libitum for 680 days. The growth of rats fed these diets
was lower than the growth of rats fed 8 mg Ca-
pantothenate/kg. The difference was significant in the
first year for the rats consuming the 2 mg Ca-

pantothenate/kg diet but not for rats consuming 4 mg
72

73

Ca-pantothenate/kg. However, after one year, weight gain
of all rats studied (consuming pantothenate supplemented
diet) plateaued and significant difference was not observed
among any groups. Weanling rats in this study on a
deficient diet for 8 weeks, weighed 40% less than rats fed
diet supplemented with 12 mg Ca-pantothenate/kg. Thus,
there is an optimal level of pantothenate required for
maximum growth of weanling rats possibly greater than 20
mg Ca-pantothenate as suggested by Henderson et a1. (1942).

Results from this study also suggest that the
requirement for pantothenic acid may be higher at the period
of growth spurt (birth to 5 weeks of age) than adulthood
of the rat. Rats fed the diet supplemented with 12 mg Ca-
pantothenate/kg ad lib excreted only 20 ug pantothenate
per 24 hours in the urine at week 2 of the study compared
to 82 ug and 72 ug per 24 hours at week 5 and 8
respectively. The ad lib control group retained in their
body 90% of the pantothenate they obtained from the diet.
Thus, during period of fast development and organ growth,
12 mg Ca-pantothenate/kg may not be sufficient for rats.
At week 5 of the study or 8 weeks of age the requirement
for pantothenate is lower and therefore the rats excreted
more pantothenate. By week 5, they excreted over four times
the amount than they excreted than week 2 while consuming
approximately the same amount of diet as week 2. At week
5 and 8, ad lib control retained 61% and 65%, respectively,

of the pantothenate consumed from diet. This was more

74
evident in rats of the deficient group. At week 2, they
only excreted 4 ug per 24 hours and remained close to that
level for the duration of the study. They received no
pantothenate from their diet; thus their body retained what
little pantothenate they had stored from birth and excreted
minimally. Rats consuming the diet supplemented with 120
mg Ca-pantothenate/kg did not experience a fluctuation in
urine pantothenate excretion since they were consuming more
than an optimal amount of pantothenate in their diet. These
rats excreted a consistent level close to 400 ug per day
at weeks 2, 5 and 8 of the study.

The question addressed is how the rats in deficient
group grew (42 grams at week 0 to 125 grams at week 8),
though at a significantly lower rate than the rats of the
ad lib control. As stated earlier, the rats consuming a
pantothenate deficient diet excreted very little
pantothenate in the urine (4 ug/24 hours from week 2 to
8). In addition very little was found in blood throughout
the study. This suggests that much of the pantothenate
remained in its respective organ.

The heart of rats in the deficient group was one organ
examined that increased in total pantothenate concentration
over time. However, total content of whole heart in rats
of the deficient group was lower than that of the ad lib
control at all weeks examined. The increase of
pantothenate concentration observed over the weeks of the

study in the deficient group rats could have come from two

75

possible sources. The heart may be siphoning pantothenate
from other organs. Spleen, for example was one organ that
decreased in pantothenate concentration in rats of the
deficient group. This decrease was not observed in rats
fed ad lib control. Muscle may have contributed much of
the pantothenate to the heart. However, pantothenate
concentration of skeletal muscle was not determined in
this study. Second possible source of pantothenic acid
may come from the feces through the practice of coprophagy.
In an unpublished study by Dr. Won Song of Michigan State
University, and also observed by Henderson et al. (1942),
the pantothenic acid content (ug/g feces) of feces was
similar in deficient pantothenic acid rats and rats fed
an adequate diet (12 mg Ca-pantothenate/kg diet).
Consumption of feces of rats in this study was reduced by
housing rats in screen bottom cages, however, coprophagy
could not be prevented. It is very possible that these
rats practiced coprophagy consistently. Barnes et a1.
(1957) reported that rats consumed 50 - 65% of excreted
feces when housed in wire bottom cages. In future studies,
it would be interesting to note any difference in
pantothenate deficient state with and without the use of
tail cups.

The prediction equation formulated was to determine
if the pantothenate level in an easily accessible body fluid
was reflective of organ pantothenate status of the rat.

Nevertheless, plasma was identified to be the best indicator

76
of pantothenate status in the rat. In a study by Cheryl
Bates of Michigan State University, she examined the
pantothenate concentration in the different blood cell
components of rats. She concluded that the total and free
pantothenate concentration in whole blood were reliable
indicator of pantothenate intake. However, she did not
examine how well correlated the blood values were to intake
nor tissue/organ level. Similarly, Eissenstat et a1. (1986)
reported that total pantothenate in fasted whole blood in
human was well correlated (r=0.38) with intake. Again,
no correlation was performed on blood concentration and
organ concentration. Predictive equations 2, 3 and 4
include whole blood, especially the free pantothenate
concentration, in the calculation of the organ (heart,
spleen and kidney) free pantothenate concentration.
However, in this study, plasma pantothenate concentration
was better correlated to the organ pantothenate
concentration than whole blood. This suggests that plasma
free pantothenate concentration is a good indicator of

pantothenate status in the rat.

CONCLUSION

Rats require pantothenate in their diets to achieve
normal growth. As demonstrated in this study, rats fed
a diet supplemented with at least 12 mg Ca-pantothenate
grew significantly better than those without pantothenate.
Those rats fed an excess of 12 mg Ca-pantothenate/kg diet,
overall, did not demonstrate greater growth than rats fed
ad lib control.

Organ pantothenate concentration was lower in rats
fed the pantothenate deficient diet compared to the control.
It is unsure from this study if the decreased level of
pantothenate concentration in the rats of the deficient
group had impaired functions. The rats fed 120 mg Ca-
pantothenate per kg of diet did not result in a higher
concentration or storage of pantothenate in their organs
compared to the control in the eight weeks of the study.

Plasma pantothenate concentration was identified as
the best indicator of organ pantothenate status. Plasma
pantothenate concentration of the rats in the deficient
group showed the highest correlation with the organ
pantothenate concentration. The formulated predictive
equations 2, 3 and 4 can be used to estimate the organ
pantothenate status of the rat. The coefficient of

77

78
determination, R2, which represents the proportion of
variation in the dependent variable (Y) explained or
associated with the variation in the independent variable,
is used as a criterion in searching for a good (predictive)
estimator. The R2 for the predictive equations were fairly

high.

LIMITATIONS

In this study, limitations were set in part by what
was feasible. All sample preparations were performed by
the principal investigator to decrease variation. It was
only possible to examine a few organs in this study with
each sacrifice. Had more organs or tissues been examined,
the speed of sample preparation by one individual would
have decreased and therefore increased the chance of
autolysis in the samples. Thus, we were selective in
choosing our "target" organs. The heart, spleen and kidney
were chosen in this study because of their role in
circulation, blood, or urine production. All RIA were
performed by the principal investigator. Precision of the
RIA was assured prior to the onset of the study. Internal
standards were used consistently with each RIA: however,
external standard (for example, yeast preparation) were
used sporadically and randomly to assure accuracy. It may
be advised to consistently used both internal and external
standard with each RIA. Behavioral changes were not
monitored in rats 24 hours a day. Perhaps videotaping the
rats may document the neurological changes associated with
human subjects deficient in pantothenate.

Only a 24 hour adaptation period was used before
starting weanling rats on the diet regimes. This was

79

80
necessary due to the rapid development of rats and for fear
of missing fast growth stage of rats. Perhaps a longer
adaptation period may be more appropriate and accurate.
A longer adjustment period will allow rats to accustom
themselves to the new environment instead of subjecting
them to more stress (of being separated from others, place
in a new cage, and fed a new diet). Regardless, all rats
were exposed to the same "stress" and differed only in diet
fed.

Diet used in this study contained 69% kcal from
carbohydrates. Fat provides 10% of kcal while protein
provides 21%. Thus, organ, blood and urine pantothenate
concentration reported from other groups may differ from
this study due to the difference in the composition of the

diet.

RECOMMENDATION

The accuracy of the predictive equation must be further
tested. In future studies, other tissues and brgans such
as skeletal muscle, lung, liver and even body pool of
pantothenate should be examined along with plasma and whole
blood concentration. The possibility of coprophagy should
also be limited. With the proper use of a tail cup, the
act of coprophagy may be limited. The use of a tail cup
will decrease consumption of intestinal synthesized
pantothenate from the feces and perhaps enhance the on-set
of pantothenate deficiency.

Pantothenate was fed as mg pantothenate per kg diet
instead of mg per kg body weight. Though this would not
have affected the deficient group, it may influence the
other pantothenate supplemented groups. The rats were
similar in body weight at the start of the study so part
the amount of pantothenate consumed per kg body weight would
have been similar. However, as the rats grew, the amount
of pantothenate consumed per unit of body weight would vary

widely.

81

 

 

 

APPENDIX

82

APPENDIX A: FOOD INTAKE OF RATS.*

Week Group A n Group B n Group C** n
1 9.7:2.4 12 10.7:1.0 12 9.1:1.2 12
2 15.911.5 12 l7.0il.4 12 9.3il.l 12
3 19.7:3.4 8 21.1:2.6 8 10.6i1.0 8
4 22.0:2.0 8 21.5:3.7 8 10.3i0.8 8
5 19.4:3.3 8 17.5:6.9 8 9.0:0.6 8
6 14.4:3.3 4 19.9:6.0 4 7.6:1.1 4
7 19.7:1.4 4 18.5:2.1 4 7.1:1.4 4
8 19.5il.5 4 l8.5:2.l 3 7.7:0.4 4

values represent mean grams of diet i stand. dev.
*Group A=rats fed 120 mg Ca-pantothenate/kg diet,
Group B=rats fed the control diet, ad lib.

ad lib.

Group C=rats fed deficient diet, ad lib.
** Same amount consumed by group C was fed to group D (pair-
fed control)

APPENDIX B: WEIGHT GAINS OF RATS.*

Week Group A n Group B n Group C n Group n
0 40:5 12 43:5 12 41:6 12 42:4 12
1 71:10 12 79:8 12 70:7 12 68:6 12
2 113:12 12 124:12 12 99:9 12 97:7 12
3 153:19 8 163:17 8 112:13 8 129:13 8
4 192:28 8 194:27 8 122:14 8 132:16 8
5 215:25 8 213:40 8 133:17 8 154:22 8
6 213:29 4 220:54 4 135:19 4 149:22 4
7 229:24 4 269:26 4 129:21 4 137:21 4
8 244:23 4 284:27 3 135:25 4 141:22 4

values represent mean grams weight : stand. dev.

*Group A=rats fed 120 mg Ca-pantothenate/kg diet, ad lib.
Group B=rats fed the control diet, ad lib.

Group C=rats fed deficient diet, ad lib.

Group D=rats pair-fed the control diet.

84

APPENDIX C: PANTOTHENATE (PA) CONCENTRATION OF RAT HEART.*

Rat I.D. Total PA Free PA Total PA Free PA
Week 2
Group A Group B
1 33.51 7.61 37.66 15.29
2 42.05 20.91 45.36 20.85
3 46.20 10.80 66.88 17.01
4 36.78 12.26 39.09 18.16
mean 39.64 12.90 47.25 17.83
std dev 5.62 5.69 13.51 2.33
Group C Group D
1 36.93 6.73 36.95 20.00
2 35.47 9.33 48.54 11.30
3 23.73 14.45 41.75 17.24
4 34.62 10.13 49.31 15.29
mean 32.69 10.16 44.14 15.96
std dev 6.04 3.21 5.87 3.66
Week 5
Group A Group B
5 36.86 20.42 51.14 19.36
6 46.18 21.63 57.17 16.02
7 41.06 17.98 48.83 15.93
8 48.65 19.93 50.22 13.97
mean 43.19 19.99 51.84 16.32
std dev 5.27 1.52 3.68 2.24
Group C Group D
5 32.80 8.19 46.85 11.02
6 31.67 10.23 51.19 14.82
7 42.92 10.96 50.80 16.43
8 42.56 18.74 52.32 16.76
mean 37.49 12.03 50.29 14.76
std dev 6.08 4.62 2.38 2.63

Appendix C continues

85

Week 8

Group A Group B
9 54.97 12.85 62.44 16.33
10 53.15 27.32 50.70 19.96
11 55.78 19.24 56.94 16.55
12 45.71 11.97 NA NA
mean 52.40 17.85 56.68 17.61
std dev 4.60 7.10 5.85 2.04

Group C Group D

9 34.38 9.94 66.72 15.45

10 57.07 8.80 47.74 18.30
11 28.49 10.97 65.08 14.18
12 38.76 14.37 50.59 21.82
mean 39.68 11.02 57.53 17.44
std dev 12.34 2.40 9.75 3.39

values represent ug pantothenate/g organ : stand. dev.
*Group A=rats fed 120 mg Ca-pantothenate/kg diet, ad lib.
Group B=rats fed the control diet, ad lib.

Group C=rats fed deficient diet, ad lib.

Group D=rats pair-fed the control diet.

86

APPENDIX D: PANTOTHENATE (PA) CONCENTRATION OF RAT SPLEEN.*

 

Rat I.D. Total PA Free PA Total PA Free PA
Week 2
Group A Group B
1 17.66 6.00 8.90 6.32
2 16.44 11.06 15.15 12.81
3 21.39 5.56 14.85 11.25
4 21.39 7.83 25.47 6.73
mean 16.78 7.61 16.09 9.28
std dev 4.04 2.50 6.88 3.25
Group C Group D
1 7.04 0.43 11.55 5.76
2 10.25 0.34 23.68 10.41
3 5.41 0.31 19.10 15.51
4 11.63 0.51 20.68 13.78
mean 8.58 0.40 18.75 11.37
std dev 2.86 0.09 5.16 4.3
Week 5
Group A Group B
5 23.28 8.51 9.44 3.13
6 15.03 6.26 12.16 1.01
7 11.90 5.32 18.10 0.51
8 12.74 3.82 22.05 2.07
mean 15.74 5.98 15.44 1.68
std dev 5.20 1.96 5.70 1.17
Group C Group D
5 10.27 1.14 12.08 2.89
6 8.35 0.70 8.90 2.32
7 5.26 0.93 9.56 2.44
8 3.67 1.99 17.35 4.84
mean 6.89 1.19 11.97 3.12
std dev 2.98 0.56 3.84 1.17

87

Appendix D continues

Week 8

Group A Group B
9 9.46 4.46 21.01 4.17
10 12.10 6.54 8.98 3.91
11 11.90 2.35 17.22 1.65
12 14.85 2.73 NA NA
mean 12.08 4.02 15.74 3.24
std dev 2.20 1.91 6.15 1.39

Group C Group D
9 3.99 0.86 8.22 5.11
10 5.02 0.92 10.64 1.40
11 4.59 1.65 5.47 5.03
12 3.46 1.25 5.27 4.95
mean 4.27 1.17 7.40 4.12
std dev 0.68 0.36 2.55 1.82

values represent ug pantothenate/g organ : stand. dev.
*Group A=rats fed 120 mg Ca-pantothenate/kg diet, ad lib.
Group B=rats fed the control diet, ad lib.

Group C=rats fed deficient diet, ad lib.

Group D=rats pair-fed the control diet.

88

APPENDIX E: PANTOTHENATE (PA) CONCENTRATION OF RAT
KIDNEY. *

Rat I.D. Total PA Free PA Total PA Free PA
Week 2

Group A Group B
1 65.80 61.30 49.01 29.32
2 84.21 40.17 54.56 34.54
3 73.58 48.71 52.82 40.13
4 65.00 41.05 63.54 30.70
mean 72.15 47.81 54.98 33.67
std dev 8.92 9.78 6.16 4.84

Group C Group D
1 33.40 20.56 59.73 38.26
2 36.95 22.86 58.25 40.83
3 39.83 20.78 30.06 27.98
4 48.93 21.24 48.01 34.92
mean 39.78 21.36 49.01 35.50
std dev 6.64 1.04 13.66 5.57

Week 5

Group A Group B
5 51.10 47.60 53.73 53.07
6 59.78 46.85 56.51 51.05
7 49.17 27.79 54.81 46.09
8 38.95 29.91 62.39 43.87
mean 49.75 38.04 56.86 48.52
std dev 8.55 10.65 3.86 4.27

Group C Group D
5 58.23 29.13 60.57 32.49
6 68.33 26.26 52.64 40.38
7 78.89 28.61 80.05 38.39
8 61.68 38.91 76.21 39.92
mean 66.78 30.73 67.37 37.80
std dev 9.10 5.60 12.94 3.64

Week 8
Group A Group B

10 57.98 55.91 55.78 47.27
11 67.90 50.33 46.95 45.03
12 66.89 38.57 NA NA
mean 64.34 49.06 53.19 44.64
std dev 4.46 7.40 5.43 2.85

89

Appendix E continues

(Week 8)
Group C Group D
9 51.37 34.14 47.15 30.66
10 40.79 33.34 42.49 29.18
11 34.76 25.86 60.93 34.51
12 41.18 28.99 61.12 25.94
mean 42.03 30.58 52.92 30.87
std dev 6.89 3.88 9.55 3.55

values represent ug pantothenate/g organ

* Group A=rats
Group B=rats
Group C=rats
Group D=rats

fed 120 mg Ca—pantothenate/kg diet, ad lib.
fed the control diet, ad lib.
fed deficient diet, ad lib
pair-fed the control diet.

90

APPENDIX F: PANTOTHENATE (PA) CONCENTRATION OF RAT WHOLE
BLOOD. *

Rat I.D. Total PA Free PA Total PA Free PA
Week 2

Group A Group B
1 1.07 0.90 1.01 0.79
2 1.66 1.19 0.43 0.27
3 1.51 1.43 0.86 0.64
4 1.36 1.21 0.71 0.32
mean 1.40 1.18 0.75 0.50
std dev 0.25 0.22 0.25 0.25

Group C Group D
1 0.32 0.24 0.50 0.41
2 0.32 0.19 0.75 0.42
3 0.32 0.24 0.68 0.35
4 0.32 0.29 0.88 0.25
mean 0.32 0.24 0.70 0.36
std dev 0.002 0.04 0.16 0.08

Week 5

Group A Group B
5 1.26 0.72 0.66 0.29
6 1.57 0.81 0.74 0.49
7 1.39 0.92 0.76 0.49
8 0.81 0.59 1.04 0.43
mean 1.26 0.76 0.80 0.43
std dev 0.32 0.14 0.17 0.09

91

Appendix F continues

(Week 5)

Group C Group D
5 0.46 0.27 0.41 0.31
6 0.39 0.25 0.66 0.46
7 0.28 0.27 0.35 0.34
8 0.47 0.36 0.54 0.45
mean 0.40 0.29 0.49 0.39
std dev 0.09 0.05 0.14 0.08

Week 8

Group A Group B
9 0.75 0.44 0.80 0.45
10 1.11 0.52 0.54 0.43
11 0.67 0.42 1.00 0.39
12 1.11 0.62 NA NA
mean 0.91 0.50 0.78 0.42
std dev 0.24 0.09 0.19 0.03

Group C Group D
9 0.56 0.26 0.80 0.40
10 0.31 0.17 0.48 0.40
11 0.36 0.25 0.54 0.47
12 0.66 0.25 0.54 0.37
mean 0.47 0.23 0.59 0.41
std dev 0.17 0.04 0.14 0.04

values represent ug pantothate/ml wh bld

*Group A=rats fed 120 mg Ca-pantothenate/kg diet, ad lib.

Group B=rats fed the control diet, ad lib.
Group C=rats fed deficient diet, ad lib.
Group D=rats pair-fed the control diet.

92

APPENDIX G: FREE PANTOTHENATE CONCENTRATION OF RAT
PLASMA. *

Rat I.D. Group A Group B Group C Group D
Week 2
1 0.86 0.26 0.02 0.04
2 1.12 0.29 0.01 0.11
3 1.08 0.32 0.02 0.08
4 0.31 0.13 0.004 0.11
mean 0.84 0.25 0.01 0.09
std dev 0.37 0.08 0.006 0.03
Week 5
5 0.56 0.30 0.29 0.34
6 0.92 0.67 0.03 0.36
7 0.83 0.47 0.04 0.22
8 0.33 0.11 0.07 0.20
mean 0.66 0.39 0.04 0.28
std dev 0.27 0.24 0.02 0.08
Week 8

9 0.45 0.32 0.02 NA
10 0.50 0.59 0.02 NA
11 0.58 0.46 0.03 NA
12 0.50 NA 0.06 NA
mean 0.51 0.46 0.03 NA
std dev 0.05 0.11 0.02 NA

values represent ug pantothenate/ml plasma

*Group A=rats fed 120 mg Ca-pantothenate/kg diet,

Group B=rats fed the control diet, ad lib.
Group C=rats fed deficient diet, ad lib.
Group D=rats pair-fed the control diet.

ad lib.

93

APPENDIX H: FREE PANTOTHENATE CONCENTRATION EXCRETED IN
URINE BY RATS. *

Rat I.D. Group A Group B Group C Group D
Week 2
1 364.30 20.40 4.62 24.49
2 379.07 17.92 3.26 29.10
3 387.19 18.16 3.33 16.03
4 360.46 23.23 4.71 16.73
mean 372.76 19.93 3.98 21.59
std dev 12.53 2.47 0.79 6.31
Week 5
5 364.31 55.39 6.51 21.38
6 369.16 71.68 2.01 52.34
7 343.68 107.19 2.62 52.62
8 343.70 93.82 3.38 98.43
mean 355.21 82.02 3.63 56.19
std dev 13.46 23.01 2.00 31.75
Week 8
9 376.09 56.53 3.92 24.14
10 378.10 103.36 4.67 17.57
11 369.14 121.13 4.74 37.93
12 327.72 NA 4.04 15.58
mean 362.76 93.67 4.34 23.81
std dev 23.67 27.25 0.42 10.10

 

 

values represent ug pantothenate excreted in urine/24 hours
*Group A=rats fed 120 mg Ca-pantothenate/kg diet, ad lib.
Group B=rats fed the control diet, ad lib.

Group C=rats fed deficient diet, ad lib.

Group D=rats pair-fed the control diet.

94

APPENDIX I: ORGAN WEIGHTS OF RATS.*

Rat I.D. Group A Group B Group C Group D

Heart (grams)

Week 2
1 0.53 0.73 0.39 0.43
2 0.54 0.50 0.46 0.44
3 0.54 0.59 0.40 0.41
4 0.53 0.96 0.41 0.39
mean 0.53 0.70 0.41 0.42
std dev 0.01 0.20 0.03 0.02
Week 5
5 0.90 0.62 0.67 0.54
6 1.02 0.86 0.61 0.48
7 0.86 0.84 0.40 0.51
8 0.70 0.71 0.53 0.49
mean 0.87 0.76 0.55 0.51
std dev 0.13 0.11 0.12 0.03
Week 8
9 1.03 0.86 0.57 0.54
10 0.94 1.01 0.73 0.54
11 0.94 1.07 0.62 0.74
12 1.02 NA 0.62 0.54
mean 0.98 0.98 0.64 0.59
std dev 0.05 0.11 0.07 0.10

Spleen (grams)

Week 2
1 0.37 0.50 0.24 0.35
2 0.40 0.42 0.30 0.32
3 0.57 0.45 0.23 0.31
4 0.44 0.45 0.29 0.34
mean 0.44 0.46 0.27 0.33
std dev 0.09 0.03 0.03 0.02
Week 5
5 0.54 0.44 0.32 0.33
6 0.62 0.62 0.28 0.28
7 0.61 0.60 0.25 0.32
8 0.54 0.35 0.27 0.27
mean 0.58 0.50 0.28 0.30
std dev 0.05 0.13 0.03 0.03

 

95

Appendix I continues
(Spleen in grams)

Week 8
9 0.56 0.55 0.25 0.24
10 0.60 0.70 0.41 0.38
11 0.54 0.48 0.31 0.54
12 0.53 NA 0.30 0.33
mean 0.55 0.57 0.31 0.37
std dev 0.03 0.11 0.07 0.13

Kidney (grams)

Week 2
1 0.94 1.36 0.89 0.88
2 1.05 1.11 1.20 0.93
3 1.08 1.16 0.81 0.46
4 1.20 0.96 0.97 0.89
mean 1.07 1.15 0.97 0.79
std dev 0.10 0.17 0.17 0.22
Week 5
5 1.56 1.27 1.17 1.12
6 1.84 1.67 1.08 0.97
7 1.62 1.75 0.93 1.08
8 1.69 1.41 1.03 0.94
mean 1.68 1.52 1.05 1.03
std dev 0.12 0.22 0.10 0.08

Week 8
9 2.06 1.68 1.05 1.08
10 1.82 2.03 1.04 1.12
11 1.91 1.90 1.60 1.52
12 1.72 NA 1.10 1.17
mean 1.88 1.87 1.20 1.22
std dev 0.14 0.17 0.27 0.20

 

*Group A=rats fed 120 mg Ca-pantothenate/kg diet, ad lib.
Group B=rats fed the control diet, ad lib.

Group C=rats fed deficient diet, ad lib.

Group D=rats pair-fed the control diet.

 

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