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Occurrence of free nicotinic acid in the liver of nicotinamide-injected rats
Vol. 17, No. 1, 1964
BIOCHEMICAL
OF FREE
OCCURRENCE
AND BIOPHYSICAL
NICOTINIC
RESEARCH COMMUNICATIONS
ACID IN THE LIVER
NICOTINAMIDELINJECTED C. Ricci Istituto
Received
di
Chimica
liver
menon (Kaplan
about
less
the
a scheme
involved
has been
accepted
that
the
mediates
(Langan
described liver,
et al.,
(Sarma to in
the
in
the
vitro
by rat
of NAc are
the
NAD syn=
This
the
that
path=
liver
of
is
NAm
NAD pro=
present
in human as inter=
NAm deamidation
at
preparations however
it
NAm conversion
or in viva.
recent=
now widely
NAc-nucleotides
liver
observa=
More
to hepatic
et a1.,1961); detect
mouse
the
and it
1959).
of
NAD biosynthetic
from
similar
involves
level
possible
for
followed
The same pheno=
promoting
NAm conversion
which
mononucleotide
rat
Siena
NAD content
doses
reported,
a pathway
erythrocytes,
the
is
et'a1.,1956).
of Des-NAD
mice
been
di
NMN as an intermediate.
isolation
through,
rat
described
NAm in
(Kaplan
injected ceeds
in
Equimolecular
than
liver
suggested the
been
1956).
effective in
IO-fold
the
and Ricci,1949).
subsequently
way which ly,
of
et al.,
thesis
of NAm to
(Bonsignore
has
tion
V. Pallini
June 25, 1964
by an increase
far
RATS
Biologiqa dell'Universit& Siena (Italy)
The administration the
and
OF
the
has been has not to
yet
NMN by
The NAm deamidase
EC=
The work was supported by Research Grant GM 0979101 from the National Institutes of Health and by C.N.R. Impresa Enzimologia. Abbreviations: NAm,Nicotinamide; NAc, acid; NAD,Wicotinamide-Adenine-Dinucleotide; tinanide Mononuoleotide; Des-NAD, Nicotinic Des-N&m, Nicotinic acid nine-Dinucleofido; tide.
34
Nicotinic NMN,Nico= acid-Ade= Mononucleo=
Vol. 17, No. 1, 1964
tivity,
BIOCHEMICAL
AND BIOPHYSICAL
reported
to be absent
previously
(Rajagopalan present
et
in
al.,
an inhibited
The inhibition
is
(Greengard
form
released
of NAc have
NAm-treated
mice
were
the
the
rat
level
reaches
a maximum
at were
strain,
were
injected
from
all
Two rats
prepared (1954).
for
in
the
a curve
which
female
laboratory wt.
100 ag of
and determined
by paper
within
were
sacrificed
acid
extracts
at from
water a short
each
time
8 g of
neutralized
extract
NAD (Holzer
et
using
procedure
a 250 ml mixing
of
interval.
al.,
was kept
a1.,1958).
6 g of liver
on a 1 x 12 cm column the
period
et
the
following
so that
was
of
to
supplied
liver
An aliquot
corresponding
the
schedule
of Hurlbert
of
pur= chromate=
After
on a time
NAm
to be free
systems.
decapitated
The
NAm was
and only
of
130
diet.
with
compounds
rats
between
procedure
mesh,
a1.(1954),
in=
the
aliquot
in
greatly
to
chromatographed 200-400
is
ranging
solvent
assay
exper= that
according
an enzymatic
A second
A.G.,
were made
Percloric
liver
on adult
was withdrawn
were
quantitative
to find
100 Q of body
two different
rats
able
of
8 th hr .
pyridine
Injections
time.
be
liver
sensitive
and follows
a standard
Merck
food
the
been
a weight
per
the
injection, freely.
to
the
but
intraperitoneally
contaminating in
in
a more
conducted
with
10% solution)
graphy
liver
hypophysectomy
NAc in
the
and 150 g : and fed
from
found
a1.,1959),
NAm injection,
Experiments
chased
rat
et a1.,1963)o
detected
Using
of free
after
Wistar
et
we have
creased
(as
(Retrack
been
lacking.
procedure,
rats
now been
after
(Langan
still
imental
the
has
in
et a1.,1963).
Traces data
1960),
RESEARCH COMMUNICATIONS
was
of Dowex 1 (HCOO-) of Hurlbert
chamber
et
and 4N HCOOH
reservoir. The NAm was washed
away from
35
the
column
with
water,
Vol. 17, No. 1,1964
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
and was determined in this fraction by the method of Hughes and Williamson (1953). As checked by chromate= graphy on Whatman 1 in the solvent C of Preiss and Handler (1958), at each time interval after injection of NAm, NAm is the tertiary pyridine compound present in the water eluate. In addition, there are slight traces of quaternary pyridine derivatives which do not interfere appreciably in the method of Hughes and Williamson. NAc was eluted from the Dowex 1 column together with NAD. In order to separate NAc from MD and its hydrolytic products, the fractions in the peak were pooled, lyophylized and taken up in 1 ml of water. Aliquots corresponding to 1.5 g of liver, were chromate= graphed on Whatman 31 paper using n-butanol:water as the descending solvent with l$ ammonia on the bottom of the jar. Under these conditions NAc migrated with a Rf of about 0 .330 The region of the paper correspond= ing to NAc, as revealed on a adjacent lane by the method of Kodiceck and Reddi (1951), was cut out and eluted with 4 ml of 0.1 N HCl with the descending technique. The eluates were brought to pH 6-7 and NAc was determined by the method of Hughes and Williamson. Under these conditions the material elutea from the paper gave the A350/A370 ratio of 0.6 which is charac= teristic of NAc. Known amounts of NAc were run through the separation procedure and NAc was recovered with a yield of 957100$. Des-NAD was eluted from the column with a higher HCOOH concentration, and was determined by its reaction with KCN (Lamborg et a1,,1958). The content of NAc of the liver at various times after NAm injection is reported in the Table I. The value shown at zero time was obtained from normal rats and is near the lower limit of the assay 36
Vol. 17, No. 1,1964
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE I NBC and other pyridine compounds in the liver of NAm treated rats. (Value5 expressed as poles/g of fresh tissue) Des-NAD NAD Hours after NAm NAc injection 0.22 0 .OOl 0.010 0.49 0 2 8' 12 16 24
5.14 5.00 4.34 1.90 1.03 0.10
0.034 0.040 0.068 0.062 0.005 0 .OOl
0.234 0.265 0.241 0.232 0.047 0.022
1.58 2.92 2.56 5.60 3.08 0.62
method. The administration of NAm is very soon followed by a rise in the NAc level and this amounts to a 70fold increase after 8 hr. The values and the time curves for the other compounds are in close agreement with the data from other authors (Langan et a1.,1959; Greengard et al. ,196l). The time curves for NAc and NAD follow a similar pattern. A sharp increase is noted iavmdiately after NAm administration, with a peak from the 8th to the 12th hr, followed by a sharp decrease to normal values. On the other hand, the curve of NAm shows an earlier maximum. It can be noted that the maximum level of free NAm around the 2nd hr almost equals the maximum level reached by NAD at the 12 th hr e The isolation of NAc from the liver of NAm-injec= ted rats further substantiates the conversion of NAm to NAD through desamidated compounds. The involvement of NAc in NAD biosynthesis is clearly indicated by the fact that its level in the liver is relatively high during the time when NAD is being synthesized. After the 12th hr it drop5 to lower values. The same can be noted for Des-NAD. The eventual accumulation of free NAc can be ex= plained by assuming that the deamidation of NAm occurs 37
Vol. 17, No. 1, 1964
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
at a higher rate than the reaction of NBC with %phos= phoribosyl-I-pgrophosphate. In fact, it has been re= cently reported by Imsande (1964) that the specific activity of rat liver Des+NMN pyrophosphorylase is relatively low as compared to that of other biological systems. We wish to thank Mr M.Bari assistance.
for
highly
skilled
REFERENCES Bonsignore A.and Ricci C.,Boll.Soc.It.Biol.Sper. 25, 710,(1949) Greengard P.,Kalinsky H.J.and Petrack Be, Biochim. Biophys.Acta %,408,(1961) Greengard P., Petrack B., Craston A. and Kalinsky H.J., Biochem.Biophys.Res~Comm.~,478,(1963) Holzer H.,Busch D. and Kroeger H., Z.Physiol.Chem. 111, 184, (1958) Hughes D.E. and Williamson D.H., Bi0chem.J. 55,851, Hurlbert
(1953)
RQB., Siohmitz H., &umm A.F. and Potter V.R., J.Biol,Chem. =,23,(1954) Imsande J., Biochti.Biophys.Acta 82,445,(1964) Kaplan N.O., Goldin A., Humphreys S.R., Ciotti M.M., and Stolzenbach F.E., J.Biol.Chem. 219, 287, (1956) Kodiceck E. and Reddi K.K., Nature m,475,(1951) Lsmborg M., Stolzenbach F.E. and Kaplan N.O., J.Biol. Chem. =,685,(1958) Langan T.A., Kaplan N.O. and Shuster Lo, J.Biol.Chem. a,2161
,(1959)
B., Greengard P., Craston A. and Kalinsky H.J., Biochem.Biophys.Res.Comm. _?1,472,(1963) =,493,(1958) Preiss J. and Handler PC, J,Biol.Chem. Rajagopalan K.V., Sarma D.S,R., Sundarsm T.K. and Sarma P.S*, Enzymologia 2&277,(1960) Sarma D.S.R., Rajalakshmi S. and Sarma P.S., Biochem. BiophyseRes.Comm. 5,389,(1961)
Petrack
38
BIOCHEMICAL
OF FREE
OCCURRENCE
AND BIOPHYSICAL
NICOTINIC
RESEARCH COMMUNICATIONS
ACID IN THE LIVER
NICOTINAMIDELINJECTED C. Ricci Istituto
Received
di
Chimica
liver
menon (Kaplan
about
less
the
a scheme
involved
has been
accepted
that
the
mediates
(Langan
described liver,
et al.,
(Sarma to in
the
in
the
vitro
by rat
of NAc are
the
NAD syn=
This
the
that
path=
liver
of
is
NAm
NAD pro=
present
in human as inter=
NAm deamidation
at
preparations however
it
NAm conversion
or in viva.
recent=
now widely
NAc-nucleotides
liver
observa=
More
to hepatic
et a1.,1961); detect
mouse
the
and it
1959).
of
NAD biosynthetic
from
similar
involves
level
possible
for
followed
The same pheno=
promoting
NAm conversion
which
mononucleotide
rat
Siena
NAD content
doses
reported,
a pathway
erythrocytes,
the
is
et'a1.,1956).
of Des-NAD
mice
been
di
NMN as an intermediate.
isolation
through,
rat
described
NAm in
(Kaplan
injected ceeds
in
Equimolecular
than
liver
suggested the
been
1956).
effective in
IO-fold
the
and Ricci,1949).
subsequently
way which ly,
of
et al.,
thesis
of NAm to
(Bonsignore
has
tion
V. Pallini
June 25, 1964
by an increase
far
RATS
Biologiqa dell'Universit& Siena (Italy)
The administration the
and
OF
the
has been has not to
yet
NMN by
The NAm deamidase
EC=
The work was supported by Research Grant GM 0979101 from the National Institutes of Health and by C.N.R. Impresa Enzimologia. Abbreviations: NAm,Nicotinamide; NAc, acid; NAD,Wicotinamide-Adenine-Dinucleotide; tinanide Mononuoleotide; Des-NAD, Nicotinic Des-N&m, Nicotinic acid nine-Dinucleofido; tide.
34
Nicotinic NMN,Nico= acid-Ade= Mononucleo=
Vol. 17, No. 1, 1964
tivity,
BIOCHEMICAL
AND BIOPHYSICAL
reported
to be absent
previously
(Rajagopalan present
et
in
al.,
an inhibited
The inhibition
is
(Greengard
form
released
of NAc have
NAm-treated
mice
were
the
the
rat
level
reaches
a maximum
at were
strain,
were
injected
from
all
Two rats
prepared (1954).
for
in
the
a curve
which
female
laboratory wt.
100 ag of
and determined
by paper
within
were
sacrificed
acid
extracts
at from
water a short
each
time
8 g of
neutralized
extract
NAD (Holzer
et
using
procedure
a 250 ml mixing
of
interval.
al.,
was kept
a1.,1958).
6 g of liver
on a 1 x 12 cm column the
period
et
the
following
so that
was
of
to
supplied
liver
An aliquot
corresponding
the
schedule
of Hurlbert
of
pur= chromate=
After
on a time
NAm
to be free
systems.
decapitated
The
NAm was
and only
of
130
diet.
with
compounds
rats
between
procedure
mesh,
a1.(1954),
in=
the
aliquot
in
greatly
to
chromatographed 200-400
is
ranging
solvent
assay
exper= that
according
an enzymatic
A second
A.G.,
were made
Percloric
liver
on adult
was withdrawn
were
quantitative
to find
100 Q of body
two different
rats
able
of
8 th hr .
pyridine
Injections
time.
be
liver
sensitive
and follows
a standard
Merck
food
the
been
a weight
per
the
injection, freely.
to
the
but
intraperitoneally
contaminating in
in
a more
conducted
with
10% solution)
graphy
liver
hypophysectomy
NAc in
the
and 150 g : and fed
from
found
a1.,1959),
NAm injection,
Experiments
chased
rat
et a1.,1963)o
detected
Using
of free
after
Wistar
et
we have
creased
(as
(Retrack
been
lacking.
procedure,
rats
now been
after
(Langan
still
imental
the
has
in
et a1.,1963).
Traces data
1960),
RESEARCH COMMUNICATIONS
was
of Dowex 1 (HCOO-) of Hurlbert
chamber
et
and 4N HCOOH
reservoir. The NAm was washed
away from
35
the
column
with
water,
Vol. 17, No. 1,1964
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
and was determined in this fraction by the method of Hughes and Williamson (1953). As checked by chromate= graphy on Whatman 1 in the solvent C of Preiss and Handler (1958), at each time interval after injection of NAm, NAm is the tertiary pyridine compound present in the water eluate. In addition, there are slight traces of quaternary pyridine derivatives which do not interfere appreciably in the method of Hughes and Williamson. NAc was eluted from the Dowex 1 column together with NAD. In order to separate NAc from MD and its hydrolytic products, the fractions in the peak were pooled, lyophylized and taken up in 1 ml of water. Aliquots corresponding to 1.5 g of liver, were chromate= graphed on Whatman 31 paper using n-butanol:water as the descending solvent with l$ ammonia on the bottom of the jar. Under these conditions NAc migrated with a Rf of about 0 .330 The region of the paper correspond= ing to NAc, as revealed on a adjacent lane by the method of Kodiceck and Reddi (1951), was cut out and eluted with 4 ml of 0.1 N HCl with the descending technique. The eluates were brought to pH 6-7 and NAc was determined by the method of Hughes and Williamson. Under these conditions the material elutea from the paper gave the A350/A370 ratio of 0.6 which is charac= teristic of NAc. Known amounts of NAc were run through the separation procedure and NAc was recovered with a yield of 957100$. Des-NAD was eluted from the column with a higher HCOOH concentration, and was determined by its reaction with KCN (Lamborg et a1,,1958). The content of NAc of the liver at various times after NAm injection is reported in the Table I. The value shown at zero time was obtained from normal rats and is near the lower limit of the assay 36
Vol. 17, No. 1,1964
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE I NBC and other pyridine compounds in the liver of NAm treated rats. (Value5 expressed as poles/g of fresh tissue) Des-NAD NAD Hours after NAm NAc injection 0.22 0 .OOl 0.010 0.49 0 2 8' 12 16 24
5.14 5.00 4.34 1.90 1.03 0.10
0.034 0.040 0.068 0.062 0.005 0 .OOl
0.234 0.265 0.241 0.232 0.047 0.022
1.58 2.92 2.56 5.60 3.08 0.62
method. The administration of NAm is very soon followed by a rise in the NAc level and this amounts to a 70fold increase after 8 hr. The values and the time curves for the other compounds are in close agreement with the data from other authors (Langan et a1.,1959; Greengard et al. ,196l). The time curves for NAc and NAD follow a similar pattern. A sharp increase is noted iavmdiately after NAm administration, with a peak from the 8th to the 12th hr, followed by a sharp decrease to normal values. On the other hand, the curve of NAm shows an earlier maximum. It can be noted that the maximum level of free NAm around the 2nd hr almost equals the maximum level reached by NAD at the 12 th hr e The isolation of NAc from the liver of NAm-injec= ted rats further substantiates the conversion of NAm to NAD through desamidated compounds. The involvement of NAc in NAD biosynthesis is clearly indicated by the fact that its level in the liver is relatively high during the time when NAD is being synthesized. After the 12th hr it drop5 to lower values. The same can be noted for Des-NAD. The eventual accumulation of free NAc can be ex= plained by assuming that the deamidation of NAm occurs 37
Vol. 17, No. 1, 1964
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
at a higher rate than the reaction of NBC with %phos= phoribosyl-I-pgrophosphate. In fact, it has been re= cently reported by Imsande (1964) that the specific activity of rat liver Des+NMN pyrophosphorylase is relatively low as compared to that of other biological systems. We wish to thank Mr M.Bari assistance.
for
highly
skilled
REFERENCES Bonsignore A.and Ricci C.,Boll.Soc.It.Biol.Sper. 25, 710,(1949) Greengard P.,Kalinsky H.J.and Petrack Be, Biochim. Biophys.Acta %,408,(1961) Greengard P., Petrack B., Craston A. and Kalinsky H.J., Biochem.Biophys.Res~Comm.~,478,(1963) Holzer H.,Busch D. and Kroeger H., Z.Physiol.Chem. 111, 184, (1958) Hughes D.E. and Williamson D.H., Bi0chem.J. 55,851, Hurlbert
(1953)
RQB., Siohmitz H., &umm A.F. and Potter V.R., J.Biol,Chem. =,23,(1954) Imsande J., Biochti.Biophys.Acta 82,445,(1964) Kaplan N.O., Goldin A., Humphreys S.R., Ciotti M.M., and Stolzenbach F.E., J.Biol.Chem. 219, 287, (1956) Kodiceck E. and Reddi K.K., Nature m,475,(1951) Lsmborg M., Stolzenbach F.E. and Kaplan N.O., J.Biol. Chem. =,685,(1958) Langan T.A., Kaplan N.O. and Shuster Lo, J.Biol.Chem. a,2161
,(1959)
B., Greengard P., Craston A. and Kalinsky H.J., Biochem.Biophys.Res.Comm. _?1,472,(1963) =,493,(1958) Preiss J. and Handler PC, J,Biol.Chem. Rajagopalan K.V., Sarma D.S,R., Sundarsm T.K. and Sarma P.S*, Enzymologia 2&277,(1960) Sarma D.S.R., Rajalakshmi S. and Sarma P.S., Biochem. BiophyseRes.Comm. 5,389,(1961)
Petrack
38