Nicotinamide nucleotides in the erythrocytes of normal human subjects and of patients suffering from pellagra were separated by paper chromatography and their concentrations determined. Though no differences were observed in the concentration of total nicotinamide nucleotides in erythrocytes of pellagrins and normals, significant differences were observed with regard to concentration of the individual nucleotides. The erythrocytes of pellagrins had significantly higher amounts of NMN than the erythrocytes of normal subjects, while levels of NAD and NADP tended to be lower in the pellagrins as compared to normals

Gopalan, C.

Narasinga Rao, B. S.

Raghuramulu, N.

Srikantia, S. G.

English

Publications of the IAS Fellows

1306
THE AMERICAN JOLRNAL OF CLINICAL NUTRITION
Vol. 21, No. 11, November, 1968, pp. 1306-1309
Printed in U.S.A.
Pattern of Nicotinamide Nucleotides in
the Erythrocytes of Pellagrins1
S. G. SRIKANTIA, B.SC., MB., B.S., D.SC.,2 B. S. NARASINGA RAO, B.SC.(H0NS), PH.D.,3
N. RAGHURAMULU, M.SC., PH.D.,4 AND C. GOPALAN, M.D., PH.D.5
T WAS REPORTED earlier from these Lab-
oratories that time concentration of nico-
tinalmmide nucleotitles in time erytimrocytes of
subjects suffering from pellagra was not
different from timat in normal subjects, but
the ability of erytimrocytes to synthesize time
Imucieotides in vitro was significantly lower
in i)ellaifls (1). It was also observed that
oral adini nistration of leucine considerably
depressed time ability of erythrocytes to
synthesize in vitro nicotinamide nucleo-
tides botim in normal subjects and in pel-
lagrins but tiid not bring about any
alterations ilm time total nucleotide concen-
tration. Tlmese apparently paradoxical re-
stIlts suggesteti that the type of nucleo-
tides in time erythrocytes of pellagrins may
be different from that in imormal subjects.
Fractionation of time nucleotides was,
therefore, carried out to examine timis pos-
sibility. In addition, time nature of nucleo-
tides svlmtimesized iim vitro in time erythro-
cytes of imornmals and pellagrins was also
examined.
MATERIALS AND METHODS
Fasting samples of vemous blood were ob-
tailmed from 12 normal subjects and 20 pella-
grms for the determination of total nicotina-
mide nucleotides and fractiolms. In 10 of these
pellagrins blood samples were again obtained
after intramuscular administration of 300 mg
1 Froln the Nutrition Research Laboratories, In-
dian Council of Medical Research, Tarnaka,
Hyderabad-7, India.
2 Deputy Director and Head of the Clinical Di-
vision. ‘Senior Research Officer. ‘Assistant Re-
search Officer. 5Director.
nicotinic acid daily for 5 days. During this pe-
nod timey received a diet timat provided daily
about 2,400 kcal, 50 g of  and 50 g of
fat.
Erythrocytes were separated, washed, and
niade up to knowim volunie with 0.1 s Ringer
pimosphate buffer, pH 7.4. To 1.5 ml of this cell
suspension, 0.5 ml of 12% perclmloric acid was
added to precipitate proteilms. After centrifuga-
tion, the supernatant was adjusted to pH 7.0
with 2 N KOH and filtered. One-tenth milliliter
of filtrate was spotted on Whatman no. 1 filter
paper and irrigated overnight in a solvent sys-
tem of seven parts of 95% ethyl alcohol and
timree parts of 1 s anmmonium acetate (pH 5.0)
by the ascending tecimnique (2). The nucleotides
were located as fluoreScent spots (3). The spots
of nicotinamide mononucleotide (NAD) and
nicotinamide adenine di nucleotide phosphate
(NADP) were identified using authentic sam-
ples. A third spot which was located above the
NAD and NADP spots and appeared only in
the erythrocytes of pellagrilms was identified as
nicotinamide mononucleotide (NMN) by its
ultraviolet absorption spectra (maxima, 264
Imme) and by paper chronmatography in a 2 : I
pyridine-water systenm. This was confirmed by
two-dimensional paper chromatography using
ammonium acetate and alcohol (R,  0.25) in the
first run and pyridine water in the second run
(R, , 04). Further, the RF value of the spot was
identical with tlmat of autlmentic samples of
NMN.
Time spots were eluted witim 1 ml of 0.01 N
HCI and estimated fluorometrically (4) using
pure samples as the standard. In the chromato-
grams of normal subjects, time area of paper cor-
responding to the NMN spot was eluted to de-
termine whether NMN was present.
It was found that 95% of the spotted quaIl-
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Erythrocytes, mg/100 ml
Number
 of Total
Subjects  NMN NAD NADP
%NMX NAD %XADP
-
NADP
nucleotides
Normals 12 5.02 ±
 0.44
2.48 ±
0.27
1.43 ±
0.21
65.42 ±
2.13
34.58 ± 1.99 ±
2.13  0.23
Pellagrins 20  4.97 ± 0.73 ± 2.13 ± 1.00 ± 18.96 ± 53.40 ± 27.63 ± 2.15 ±
0.66 0.09 0.19 0.17 2.11 2.53 2.45  0.22
Pellagrins after 10 4.97 ± 0.18 ± 3.01 ± 1.37 ± 6.50 ± 62.80 ± 30.70 ± 2.18 ±
treatment 0.66 0.04 0.45 0.14 2.04 2.79 2.45  0.21
a Not detectable except in three subjects.
a Average of three determinations. Nucleotides
were separated by paper chromatograph. Details
given in text. Conditions of incubation have been
described earlier (Raghuramulu et al. (1)).
N icotinamide Nucleotides in Pellagra 1307
II).
TABLE I
Fractionation of nicotinamide nucleotides in the erythrocytes of norlmmals and pellagrins
Values are means ± SE.
tity was eluted out by this method and that as
low as 1 j.tg of the nucleotide could be estimated.
Time recovery of the added authentic sample to
time filtrate was 90%.
RESULTS
The amounts of time various nucleotides
present in time erythrocytes of normal sub-
jects and of pellagrins are given in Table
I. The erythrocytes of pellagrins had con-
siderable amounts of NMN, which were
not present in the erythrocytes of most
normal subjects. it was, however, present
to the extent of 2-5% of total nucleotides
in three subjects. Levels of NAD and
NADP in the erytlmrocytes of pellagrins
were lower than in normal subjects, but
these differences were not statistically sig-
nificant. The ratio of NAD to NADP was
similar in both time groups. Administration
Of nicotinic acid to pellagrins brought
about a significant reduction in NMN con-
centration and an increase in both the
NAD and NADP levels. These increases,
however, were not statistically significant.
Total nicotinamide nucleotide concentra-
tion remained unchanged after treatment.
Fractionation of nucleotides formed dur-
ing in vitro incubation of the erythrocytes
(1) indicated timat almost all of the newly
synthesized nucleotides were NAD, both in
the normal and in time pellagrin (Table
TABLE II
Separation of nicotinamide nucleotides of
erythrocytes before and after
in vitro synthesis
Erythr ocytes, mg/ 100 ml’
NMN  NAD
2 .5
10.4
1 .8
5.4
NADP

1 .4
1 .5
 0.9
 1.1
Normal subjects
Before synthesis
After synthesis
Pellagrins
Before synthesis
After synthesis
0. 10 

0.8
1 .0 
DISCUSSION
The nucleotides separated on paper ac-
counted for 75% of time estinmated total
nucleotides in the normal subjects as well
as in time pellagrins. The alkali-acetone
condensation method (3) employed here
estimated all the N-substituted nicotin-
amide compounds. However, the spots
identified and eluted from the paper chro-
matograms may not account for all the
nucleotides because nucleotides other than
NAD and NADP present in amounts in-
sufficient to be visualized as spots would
not be estimated by this procedure. The
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2
- NAD
NAD Pyrophosphorylase
1308 Srikantia et al.
Nicotinamide  NMN
V
4 
Nicotinic acid -> NAMN
SCHEME I. Sonme of the reactions involved in the synthesis and breakdown of NAD.
values of NAD and NADP obtaineti in
norimmal subjects in time present investiga-
tion are in close agreement to those re-
ported iy otimer workers using different
methods (5-7).
l’he significant observation made imere is
that though the total nucleotide content
of ei-ythi-ocytes of pellagrins is similar to
that of imorinal subjects, time Proportion of
the different nucleotides is considerably
different. Erythiocytes of pellagrins were
fouimd to contain significantly higher
amounts of NMN anti lower amounts of
NAD an(1 NADP, as compared to the
erytimrocytes of nornmal subjects.
Iimcreased amounts of NMN in time eryth-
rocytes of peilagrins may be due to one of
several reasons. Some of the reactions tlmat
are involved in the synthesis of NAD are
indicated im Scimeme 1 . Nicotiimamide inon-
onucieotide may accunmulate because of
a) an accelerated rate of conversion of nico-
tinamide to NMN (reaction 1), b) a slower
rate of conversion of NMN to either NAD
(reaction 2) or to NAMN (reaction 4), or
c) an accelerated rate of breakdown of
NAD to NMN (;eaction 3). It has been
denmonstrated that nicotinamide can serve
as a precursor for time in vitro synthesis of
NMN by human erytimrocytes (8), but time
Km value for nicotinamide is very high,
suggesting thereby that NMN is not a
physiological intermediate in the biosyn-
thesis of NAD (2). Similarly, time equilib-
riunm of tlme reaction between NMN and
and NAD is sucim timat it contributes more
to time breakdown of NAD than to its syn-
thesis (9). The NMN formed as a result of
NAD breakdown, however, can be reuti-
lized for NAD synthesis by conversioim into
nicotinic aciti mononucleotide (NAMN)
by time action of NMN deamidase (10). Nic-
otinamide mononucleotide deami(Iase has
been showim to be present in immammahan
liver, but its presence in huimman erythro-
cytes has still to be established.
The observation made here that levels
of NAD are lower in time erythrocytes of
pellagrins than in nornmals strongly sug-
gests timat time iimcreased amoulmts of NMN
are due to enimanced breakdown of NAD.
The demonstrated lowered capacity of
erytimrocytes of peliagrins to synthesize
NAD (1) anti time accumulation of NMN
in timese erytimrocytes may intleeti be re-
lated. Wimether lowered activity of NMN
deamidase is also involved, tlitis affecting
the reutilization of NMN for NAD syn-
thesis, has to await time tienmonstration of
the presence of timis enzyme in human
erythrocytes. Timese aspects are now cur-
rently being studied.
SUMMARY
Nicotinamitle nucleotides in time erythro-
cytes of normal Imuman subjects anti of pa-
tients suffering from pellagra were sepa-
rated by paper clmromatography and their
concentrations determi neti. Though no
differences were observed in time concentra-
tion of total nicotinamide nucleotides in
erythrocytes of pellagrins and normals,
significant differences were observed witim
regarti to concentration of time individual
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N icotinamide Nucleotides in Pellagra 1309
nucleotides. Time erytimrocytes of peliagrins
imad significantly higher amounts of NMN
timan time erytimrocytes of norimmal subjects,
wlmile levels of NAD and NADP tended to
be lower in the peliagrins as coimmpared to
normals.
REFERENCES
1. RAGHURAMULU, N., S. G. SRIKANTIA, B. S. NARA-
SINCA RAO AND C. GOPALAN. Nicotinarnide imu-
cleotides in the erythrocvtcs of patients suffering
from pellagra. Biochem. J. 96: 837, 1965.
2. PREI5S, J., AND P. HANDLER. Biosynthesis of di-
l)ll05Ph0-PYridine nucleotide. I. Identification of
illtcrnsc(liates. 1- Biol. Chem. 233: 488, 1958.
3. KODICEK, E., AND K. K. REDDI. Paper chronmatog-
raphy of nicotinic acid derivatives. Nature 168:
475, 1931.
4. Irvjrs, N., J. ROBINSON, F. ROSEN, J. W. HUFF
AND %V. A. PERZSVEIG. The fluorescent condensa-
lion product of N’-methyl nicotinamide and
acctone. III. A rapid fluorometric method for
the (letcrnhinatiolm of the total pvridine nuclco-
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169, 1947.
5. BISHOP, C., M. RANKINE AND J. H. TALBOTT. The
nucleotides in normal hunman blood. J. Biol.
C/tern. 234: 1233, 1959.
6. MILLS, G. C., AND L. B. SUMMERS. Time metabo-
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Arc/i. Biochern. Biop/iys. 84: 7, 1959.
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Pyridine nucleotides in erythrocyte metabolism.
J. Gun. Invest. 45: 249, 1966.
8. LEDER, I. G., AND P. HANDLER. Synthesis of
nicotilmamide mononucleotide by human eryth-
rocytes in vitro. J. Biol. C/zero. 189: 889, 1951.
9. KORNBERG, A., AND W. E. L. PRICER, JR. Enzy-
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10. SARMA, P. S. Interrelationship between trypto-
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Pattern of nicotinamide nucleotides in the erythrocytes of pellagrins

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Pattern of nicotinamide nucleotides in the erythrocytes of pellagrins

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