J Nutr Sci Vitaminol, 59, 509–515, 2013

Pantothenic Acid Deficiency May Increase the Urinary Excretion

of 2-Oxo Acids and Nicotinamide Catabolites in Rats

Katsumi Shibata, Kasumi Inomoto, Chifumi Nakata and Tsutomu Fukuwatari

Department of Nutrition, School of Human Cultures, The University of Shiga Prefecture,
Hikone, Shiga 522–8533, Japan
(Received June 20, 2013)

Summary  Pantothenic acid (PaA) is involved in the metabolism of amino acids as well as
fatty acid. We investigated the systemic metabolism of amino acids in PaA-deficient rats. For
this purpose, urine samples were collected and 2-oxo acids and l-tryptophan (l-Trp) and its
metabolites including nicotinamide were measured. Group 1 was freely fed a conventional
chemically-defined complete diet and used as an ad lib-fed control, which group was used
for showing reference values. Group 2 was freely fed the complete diet without PaA (PaA-
free diet) and used as a PaA-deficient group. Group 3 was fed the complete diet, but the daily
food amount was equal to the amount of the PaA-deficient group and used as a pair-fed con-
trol group. All rats were orally administered 100 mg of l-Trp/kg body weight at 09:00 on
day 34 of the experiment and the following 24-h urine samples were collected. The urinary
excretion of the sum of pyruvic acid and oxaloacetic acid was higher in rats fed the PaA-free
diets than in the rats fed pair-fed the complete diet. PaA deficiency elicited the increased uri-
nary excretion of anthranilic acid and kynurenic acid, while the urinary excretion of xanth-
urenic acid decreased. The urinary excretion of l-Trp itself, 3-hydroxyanthranilic acid, and
quinolinic acid revealed no differences between the rats fed the PaA-free and pair-fed the
complete diets. PaA deficiency elicited the increased excretion of N1-methylnicotinamide,
N1-methyl-2-pyridone-5-carboxamide, and N1-methyl-4-pyridone-3-carboxamide. These
findings suggest that PaA deficiency disturbs the amino acid catabolism.
Key Words  pantothenic acid, tryptophan, nicotinamide, 2-oxo acid, urine

We have studied factors affecting the amino acid
l-tryptophan (l-Trp)-nicotinamide (Nam) metabolism
(1–5). The roles of vitamins B2 and B6 are well known
from the early period in the catabolism of l-Trp to Nam
(6–9). The complete clarification of l-Trp-Nam pathway
was completed by the development of measurement of
the end Nam catabolites, N1-methyl-2-pyridone-5-car-
boxamide (2-Py) and N1-methyl-4-pyridone-3-carbox-
amide (4-Py) in 1988 (10). The method first made it
possible to calculate the conversion efficiency of l-Trp
to Nam in rats since the amount of 4-Py occupies over
80% of the Nam and its catabolites in rats (11). We
already reported the effects of vitamin B1 (12), B2 (13),
and B6 (14) deficiencies on the conversion ratio of l-Trp
to Nam in rats.
Pantothenic acid (PaA) is a member of the B-group
vitamins. which are integratively and cooperatively con-
cerned with the metabolism of fatty acids and amino
acids. We reported that PaA deficiency causes the accu-
mulation and re-administration of PaA decreases in the
tissue fat in rats fed the PaA-free diet (15). Until now,
many reports have been published about the effects
of PaA deficiency, for example, retarded growth (16),
reduced adrenal function (17), severe anatomical and
functional impairments of the testes with resulting loss
of fertility (18, 19), high serum levels of triglyceride and
E-mail: kshibata@shc.usp.ac.jp
fatty acids (20), and suppression of the proliferation
and promotion of the differentiation of keratinocytes
(21). In addition, Trulson and Ulissey (22) reported
that low doses of l-Trp are lethal in rats with adrenal
insufficiency, which implies the involvement of PaA in
the metabolism of l-Trp. PaA is involved in the impor-
tant catabolic reactions of 2-oxo acids derived from
amino acids by the deamination reaction, for example,
pyruvic acid, 2-oxoglutaric acid, 2-oxoadipic acid, and
2-oxo-branched acids. We recently developed the mea-
surement of 2-oxoadipic acid (23), which is a common
catabolite of l-Trp and l-lysine.
In the present study, we investigated how 2-oxo acids
(Fig. 1) and l-Trp metabolism (Fig. 2) are affected by
PaA-deficient status.
MATERIALS AND METHODS
Chemicals.  Casein from vitamin-free milk, l-methi-
onine, and sucrose were purchased from Wako Pure
Chemical Industries, Ltd. (Osaka, Japan). Corn oil was
obtained from Ajinomoto (Tokyo, Japan). A mineral
mixture (AIN-93-G-MX) (24) and vitamin mixture
(AIN-93-VX) (24) were purchased from Oriental Yeast
Co., Ltd. (Tokyo, Japan).
l-Trp, anthranilic acid (AnA), quinolinic acid (QA),
calcium pantothenate, pyruvic acid, oxaloacetic acid,
2-oxoglutaric acid, and 2-oxoadipic acid were purchased
from Wako Pure Chemical Industries.

509
510 Shibata K et al.

Thr

Met Gly
Phe
Trp Lys Ser
Cys

Tyr
Ala
2-Oxoadipic acid

p-Hydroxyphenyl- Pyruvic acid

Pyruvic acid
Acetyl-CoA
Citric acid Arg
2-Oxoisocaproic

Gln
Leu
Oxaloacetic acid Pro
acid

Glu

2-Oxoglutaric acid

Asp
His
Succinyl-CoA

Asn
2-Oxoiso- 2-Oxo-3-methyl-
valeric acid valeric acid

Val Ile

Fig. 1.  2-Oxo acid metabolism. Urinary excretion amounts of pyruvic acid, oxaloacetic acid, 2-oxoglutaric acid, and
2-oxoadipic acid were measured.

Xanthurenic acid (XA), kynurenic acid (KA) and
3-hydroxyanthranilic acid (3-HA), and N1-methylnic-
otinamide (MNA) chloride were purchased from Tokyo
Chemical Industry Co., Ltd. (Tokyo, Japan). N1-Methyl-
2-pyridone-5-carboxamide (2-Py) and N1-methyl-
4-pyridone-3-carboxamide (4-Py) were synthesized by
the methods of Pullman and Colowick (25) and Shibata
et al. (10), respectively. All other chemicals used were of
the highest purity available from commercial sources.
Animals and diets.  The care and treatment of the
experimental animals conformed to the University of
Shiga Prefecture guidelines for the ethical treatment of
laboratory animals. The room temperature was main-
tained at around 22˚C and about 60% humidity with
a 12/12 light/dark cycle (06:00–18:00/18:00–06:00).
Male 3-wk-old Wistar rats obtained from CLEA Japan,
Inc. (Tokyo) were divided into three groups (n55 per
group) and each rat was housed in a metabolic cage
CT-10 (CLEA Japan). Group 1 was freely fed a conven-
tional chemically-defined complete diet (Table 1) and
used as an ad lib-fed control. Group 2 was freely fed the
complete diet without PaA (PaA-free diet) (Table 1) and
used as a PaA-deficient group. Group 3 was fed the com-
plete diet (Table 1), but the daily food amount was equal
to the amount of the PaA-deficient group and the group
was used as a pair-fed control group. The experimental
period was for 34 d. The body weights and food intakes
were measured daily at around 09:00. The last day of
the experiment (09:00 at day 34), each rat was orally
administered 100 mg l-Trp/kg body weight dissolved
in saline, in order to strengthen the effects of PaA-
deficiency on the metabolism of l-Trp. The 24-h urine
samples were collected at 09:00 (day 34)–09:00 (day
35) of the last day in amber bottles containing 1 mL of
 Amino Acid Metabolism in Pantothenic Acid Deficient Rats 511

Body protein Daily Trp intake

turnover
Serotonin
5-HIAA

Protein N-Formylkynurenine Melatonin

synthesis

Kynurenine
AnA KA

3-Hydroxy-
kynurenine

XA
NaAD

ACMS

NaMN NiA
3-HA

2-Amino- AMS
muconic
acid

Fig. 2.  l-Trp metabolism. Urinary excretion amounts of l-Trp, AnA, KA, XA, 3-HA, QA, Nam, MNA, 2-Py, and 4-Py
were measured. AnA, anthranilic acid; KA, kynurenic acid; XA, xanthurenic acid; 3-HA, 3-hydroxyanthranilic acid; QA,
quinolinic acid; Nam, nicotinamide; MNA, N1-methylnicotinamide; 2-Py, N1-methyl-2-pyridone-5-carboxamide; 4-Py,
N1-methyl-4-pyridone-3-carboxamide.

Table  1.  Compositions of the diets. was tested by using Student’s two-tailed t-test. The dif-
ferences p,0.05 were considered to be statistically sig-
Complete diet PaA-free nificant. The values of ad-lib control were shown as ref-
(Control) diet erence values. GraphPad Prism (version 5.00; obtained
from GraphPad Software, Inc., San Diego, CA) was used
g/kg diet
for statistical analysis.
Vitamin-free milk casein 200 200
l-Methionine 2 2 RESULTS
Sucrose 703 703
Corn oil 50 50 Nutritional variables
Mineral mixture (AIN-93-G-MX)1 35 35 Figure 3 shows the daily changes in food intakes and
Vitamin mixture (AIN-93-VX)1 10 0 body weight in weaning rats fed the complete diet (ad-lib
PaA-free vitamin mixture (AIN-93)1 0 10 fed and pair-fed groups) and PaA-free diet (PaA-deficient
1 
group). The data on the ad-lib fed control are shown as
Reeves (24).
reference values. The food intake in rats fed the PaA-free
diet became lower from day 5 compared that in rats fed
the complete diet. After that, the food intake stayed at
1 mol/L HCl and were stored at 225˚C until needed. around 10 g/d. Nevertheless, the body weight in rats fed
Analyses of urine.  The urine amounts of Trp (26), the PaA-free diet gradually increased as shown in Fig.
5-HT (27), 5-HIAA (27), AnA (28), KA (29), XA (30), 3B. The body weight gain was lower in rats fed the PaA-
3-HA (30), QA (31), Nam (10), MNA (32), 2-Py (10), free diet than in rats pair-fed the complete diet.
4-Py (10) and 2-oxoadipic acid (23) were measured as The urinary excretion of PaA on the last day of the
described. The pyruvic acid, oxaloacetic acid, and 2-oxo- experiment is shown in Fig. 4A. The excretion amount
glutaric acid were reacted with DMB, derivatized to fluo- of rats in the PaA-deficient group was almost zero.
rescence compounds, and measured by HPLC (23). Urine 2-oxo acids
Statistical analysis.  For the statistical evaluation, CoA is an essential compound for the metabolism of
the significance of the differences in the mean values 2-oxo acids. We measured the urinary excretion of 2-oxo
between the pair-fed control and PaA-deficient groups acids such as pyruvic acid, oxaloacetic acid, 2-oxoglu-
512 Shibata K et al.

25 300
A B
20 250

Food intake (g/d)

Body weight (g)

200
15
150
10 *
100
5 50

0 0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
d d

Fig.  3.  Effect of PaA deficiency on the food intake (A) and body weight gain (B). Weaning male rats of the Wistar strain,
3 wk old, were divided into three groups: Group 1 () was freely fed a conventional chemically-defined complete diet
(Table 1) and used as an ad lib-fed control. Group 2 () was freely fed the complete diet without PaA (PaA-free diet) (Table
1) and used as a PaA-deficient group. Group 3 () was fed the complete diet (Table 1), but the daily food amount was equal
to the amount of the PaA-deficient group and the group was used as a pair-fed control group. The experimental period
was for 34 d. The body weights and food intakes were measured daily at around 09:00. Values are means6SE for 5 rats.
The significance of the differences in the mean values at the last day between the pair-fed control and PaA-deficient group
was tested by using Student’s two-tailed t-test. The differences p,0.05 were considered to be statistically significant from
the pair-fed control.

1000
A Urine pyruvate+oxaloacetate 150
B
Urine PaA (nmol/d)

800 (arbital unit)

600 100
*
400
50
200
*
0
Ad lib-fed Pair-fed PaA-def 0
Ad lib-fed Pair-fed PaA-def

50
Urine 2-oxoglutaric acid

C 2.5
D
Urine 2-oxoadipic acid

40
2.0
( mol/d)

30
( mol/d)

1.5

20
1.0

10
0.5

0
Ad lib-fed Pair-fed PaA-def 0.0
Ad lib-fed Pair-fed PaA-def

Fig.  4.  Effect of PaA deficiency on the urinary excretion amounts of PaA (A), sum of pyruvic acid and oxaloacetic acid (B),
2-oxoglutaric acid (C), and 2-oxoadipic acid (D). Values are means6SE for 5 rats. The significance of the differences in the
mean values at the last day between the pair-fed control and PaA-deficient group was tested by using Student’s two-tailed
t-test. * The differences p,0.05 were considered to be statistically significant from the pair-fed control.

taric acid, and 2-oxoadipic acid to investigate systemic olism of l-Trp-degradation. The data on the ad-lib fed
metabolic disturbance of amino acids. The data on the control are shown as reference values. PaA deficiency
ad-lib fed control are shown as reference values. Since elicited increased urinary excretion rates of AnA and
we could not separate pyruvic acid and oxaloacetic acid, KA, while the urinary excretion rate of XA decreased, as
the values of the two compounds are shown together shown in Table 2. No differences in the urinary percent-
(Fig. 4B), and were higher in the PaA deficient rats than ages of l-Trp itself, 3-HA, or QA were observed between
in the pair-fed rats. No differences in the urinary excre- the rats fed the PaA-free and pair-fed complete diets
tory amounts of 2-oxoglutaric acid and 2-oxoadipic acid (Table 2).
were observed between the PaA deficient and pair-fed Urine Nam and its catabolites
rats (Fig. 4C and D). We investigated how PaA deficiency affects the Nam
Urine metabolites of l-Trp→QA catabolism. The data on the ad-lib fed control are shown
We investigated how PaA deficiency affects the metab- as reference values. In the present experiment, the diets
 Amino Acid Metabolism in Pantothenic Acid Deficient Rats 513

Table  2.  Urinary excretion percentages of l-Trp itself and its metabolites.

l-Trp AnA KA XA 3-HA QA

(%) (%) (%) (%) (%) (%)

Ad-lib fed 0.02360.010 0.11460.011 0.47760.020 0.37460.012 0.05660.008 1.93360.327

Pair-fed 0.03660.010 0.08360.007 0.33060.028 0.27860.027 0.06060.007 0.29560.035
PaA-deficient 0.03560.010 0.12960.011* 0.48560.049* 0.15660.017* 0.07860.008 0.31560.067

Values are means6SE for 5 rats. The significance of the differences between the pair-fed control and PaA-deficient group
was tested by using Student’s two-tailed t-test. * The differences p,0.05 from the pair-fed control were considered to be
statistically significant.

Table  3.  Nutritional variables in rats at the final day of the experiment.

l-Trp intake from food Orally administered l-Trp Total intake of l-Trp NiA intake
(mmol/d) (mmol/d) (mmol/d) (mmol/d)

Ad lib-fed 21569 12864 34366 4.660.1

Pair-fed 12365 7761 20061 2.760.1
PaA-deficient 118614 6762* 185615 2.560.2

Values are means6SE for 5 rats. The significance of the differences between the pair-fed control and PaA-deficient group
was tested by using Student’s two-tailed t-test. * The differences p,0.05 from the pair-fed control were considered to be
statistically significant.

Table  4.  Urinary excretion amounts of Nam and its metabolites, and the excretion ratios.

Nam MNA 2-Py 4-Py Sum

4-Py/2-Py (2-Py14-Py)/MNA
(nmol/d) (nmol/d) (nmol/d) (nmol/d) (mmol/d)

Ad-lib fed 224677 9356134 9826118 8,7706458 10.960.7 9.4361.18 11.261.6

Pair-fed 2969 173613 11365 1,1166103 1.460.1 9.8060.56 7.260.7
PaA-deficient 35617 1,1216226* 316649* 2,2176159* 3.760.4* 7.3760.61* 2.961.0*

Values are means6SE for 5 rats. The significance of the differences between the pair-fed control and PaA-deficient group
was tested by using Student’s two-tailed t-test. * The differences p,0.05 from the pair-fed control were considered to be
statistically significant.

contained the preformed niacin, nicotinic acid. The
intakes on the last day are shown in Table 3. There-
fore, Nam and its catabolites originate not only from
l-Trp but also nicotinic acid. PaA deficiency elicited the
increased excretion of MNA, 2-Py, and 4-Py, and there-
fore, the sum of Nam and its catabolites, as shown in
Table 4. The ratios of 4-Py/2-Py and (2-Py14-Py)/MNA
were lower in the rats fed the PaA-free diet than in rats
fed pair-fed the complete diet (Table 4).
DISCUSSION
PaA involved in the metabolism of amino acids such
as CoA as well as fatty acids. PaA deficiency caused the
retardation of the body weight gain of growing rats
compared to the pair-fed control group. The retardation
of body weight gain means a shortage of energy intake.
As the energy intakes for the PaA-deficient group and
pair-fed group were exactly the same, the retardation in
the PaA-deficient rats suggests the inhibition of diges-
tion and absorption of macronutrients.
We investigated how PaA deficiency disturbs the
metabolism of amino acids. CoA is the member of 2-oxo
acid dehydrogenases, such as pyruvic acid, 2-oxoglu-
taric acid, and 2-oxoadipic acid. Therefore, PaA-defi-
cient rats must eliminate an extremely large accumu-
lated amount of 2-oxo acids in the body into urine.
However, the urinary excretion amounts of 2-oxoglu-
taric acid and 2-oxoadipic acid were almost the same for
the PaA-deficient and the pair-fed control rats. The uri-
nary amounts of both groups were 1/2 that of the ad-lib
fed control, which was proportional to the food intake.
Thus, the urinary excretion amounts of 2-oxoglutaric
acid and 2-oxoadipic acid are controlled by the intakes
of the related amino acids. On the other hand, the uri-
nary excretion of the sum of pyruvic acid and oxalo-
acetic acid was higher in the PaA-deficient rats than
in the pair-fed control rats. We could not separate the
two compounds (23); thus we showed their values as a
sum. Pyruvic acid is produced not only by amino acids
such as l-alanine but also by glucose. In this connec-
tion, l-alanine is a catabolite of l-Trp, which is produced
by the reaction of l-kynurenine→AnA1l-alanine and
514 Shibata K et al.
l-3-hydroxykynurenine→3-hydroxyanthranilic acid1l-
alanine. The urine amount of the sum of pyruvic acid
and oxaloacetic acid in the pair-fed control rats was 1/2
that of the ad-lib control rats, which was proportional
to the food intake. This was not the case for the urine
amount of the sum of pyruvic acid and oxaloacetic acid
in the PaA-deficient rats; no difference in the urinary
excretion of the sum of pyruvic acid and oxaloacetic
acid was observed, although the intake of food in the
PaA-deficient rats was 1/2 that of the ad-lib fed rats.
This finding means that PaA deficiency elicits the inhi-
bition of the reaction of pyruvic acid dehydrogenase,
which leads the accumulation of pyruvic acid. Leoni
et al. (33) reported that lactic acid is elevated in PaA
kinase-associated neurodegeneration patients. We did
not measure lactic acid, which is a metabolite of pyruvic
acid. To systematically explore the effects of PaA defi-
ciency, it is better to measure the hydroxyl acids derived
from 2-oxo acids.
PaA is not directly involved in the l-Trp catabolic
pathway, but it is concerned with the metabo­ lism;
for example, pyruvic acid is produced by the reac-
tions of l-kynurenine→AnA1l-alanine and l-3-
hydroxykynurenine→3-HA1l-alanine, both of which
are catalyzed by the same enzyme, PLP-dependent
enzyme kynureninase. PaA deficiency elicited increased
urinary excretion percentages of AnA and KA, while
the urinary excretion percentages of XA decreased. KA
and l-Ala are formed from l-kynurenine1pyruvic acid
by PLP-dependent enzyme kynurenine aminotransfer-
ase (34) and XA and l-glutamic acid (l-Glu) are formed
from l-3-hydroxykynurenine12-oxoglutaric acid by
kynurenine aminotransferase (34). PaA deficiency
disturbed the metabolism of kynurenine; PaA elicited
the inhibition of the reaction of l-kynurenine→l-3-
hydroxykynurenine, which is catalyzed in the pres-
ence of NADPH by FAD-dependent enzyme kynurenine
3-monohydroxylase (35). The integrated cooperation
of CoA, PLP, FAD, and NADPH is not known, but such
cooperation is plausible. Future studies must elucidate
the interesting, complicated, and sophisticated interre-
lationship of B-group vitamins.
We analyzed the effects of PaA deficiency on the
catabolism of Nam. In the present experiment, we used
diets containing preformed niacin, nicotinic acid, so that
we cannot calculate the conversion percentage of l-Trp
to Nam. This is for preventing niacin deficiency; if PaA
deficiency causes a decrease in the conversion of l-Trp
to Nam, PaA deficient rats also fall into niacin deficiency.
So, we compared the absolute urinary excretion of Nam
and its metabolites between the PaA-deficient rats and
pair-fed control rats. The urinary excretion amounts
of Nam catabolites such as MNA, 2-Py, and 4-Py were
higher in the PaA-deficient rats than in the pair-fed
control rats. The urinary excretion ratio (2-Py14-Py)/
MNA was lower in the PaA-deficient rats than in the
pair-fed control rats, which indicates that the reaction
of MNA→2-Py and 4-Py is lower in the PaA-deficient
rats than in the pair-fed control rats. In other words, the
activities of 2-Py-forming MNA oxidase and 4-Py-form-
ing MNA oxidase were lower in the PaA-deficient rats
than in the pair-fed control rats. The extremely higher
urine excretion of MNA in the PaA-deficient rats is its
evidence. The ratio (2-Py14-Py)/MNA reflects amino
acid nutritional status (36–38). Thus, PaA deficiency
elicits a worsening of amino acid nutritional status.
Another urine ratio, 4-Py/2-Py, was lower in the PaA-
deficient rats than in the pair-fed control rats, indicating
that PaA deficiency lowered the activity of 4-Py-forming
MNA oxidase rather than the activity of 2-Py-forming
MNA oxidase. This effect of PaA deficiency might not be
a direct action but an indirect action through worsening
amino acid nutrition.
In conclusion, our study which used urine samples,
illustrates the value of metabolic profiling as a tool for
systematically exploring the biochemical basis of the
PaA-deficient state.
Acknowledgments
This investigation was part of the project “Stud-
ies on the nutritional evaluation of amino acids and
B-group vitamins” (principal investigator, Katsumi Shi-
bata), which was supported by a Grant-in-Aid for Scien-
tific Research from Japan Society for the Promotion of
Science.
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