Download `Metabolic Pool` and the Use of 15N-Labelled

349
Clinical Science (1981) 61,349-35 1
CORRESPONDENCE SECTION
‘Metabolic pool’ and the use of lSN-labelledamino acids
M. JEEVANANDAM,
Department of Surgery, College of Physicians and Surgeons,
Columbia University, 630 West 168th St, New York, N Y 10032, U.S.A., 3 November 1980
The paper in Clinical Science (1980) 58, 517522 by Jackson & Golden 111 gives a somewhat
misleading idea of the conception of a ‘metabolic
pool’ and usefulness of the stable isotope of
nitrogen, 15N, in the investigation of nitrogenexchange kinetics.
In the first sentence, with a wrong reference [21
they have stated that ‘the model of a single
functional metabolic pool of cc-amino nitrogen
has been useful in the conception of nitrogen
metabolism in the body’. Actually, Sprinson &
Rittenberg 131 had defined the ‘metabolic pool’ as
that mixture of compounds derived either from
the diet or from the breakdown of tissues that the
body employs for the synthesis of tissue constituents. Their second reference [4] did not single
out the ‘metabolic pool’ of nitrogen as that solely
of cc-amino nitrogen. The mobility of the amide
nitrogen of glutamine cannot be ignored in
considering the body nitrogen exchange kinetics.
In defining the overall character and size of a
‘metabolic pool’ of nitrogen in the body one
cannot isolate transaminating amino acids from
deaminating ones.
For the investigation of nitrogen mobilization
in the whole body, the concept of a single
‘metabolic pool’ is obviously an over simplification. The original method [31 had been
modified and extended. Lately, in addition to P N I
glycine, various 15N-labelled amino acids like
alanine, lysine, phenylalanine and aspartic acid
had been used in humans and they all give invariably the same order of rate of protein turnover within a factor of 2 to 3, suggesting that the
‘metabolic pool’ handles the isotope in a somewhat similar manner irrespective of the source of
its origin. It is well known that all amino acids do
not take part to the same extent in nitrogen
exchange 151. The size of the ‘active metabolic
pool’ corresponds only to about 12% of the total
free amino nitrogen pool in the whole body of a
70 kg normal man [61, suggesting that only a
fraction of the total body amino nitrogen takes an
active part in nitrogen metabolism. The metabolic
0143-5221/8 1/090349-03$01.50/1
pool is ‘a complex mixture of compounds’, the
size of which may be affected by the diet, disease
and other factors. It is not solely of ‘the a-amino
nitrogen pool as classically conceived’ and does
not exclude fully the five amino acids specified by
the authors.
They have measured the 15N content of
alanine, glycine and glutamate of plasma and
urea and ammonia of urine samples taken during
a continuous infusion of [l5N1 glycine in two
normal adult subjects. However, in three of the
four studies, the system was disturbed during the
isotope infusion by nutritional intervention. The
reported conclusion that the nitrogen of glycine is
not transferred to alanine in any of the metabolic
states, was based on a poorly designed experimental protocol for this purpose and a questionable method of analysis. The uncertainty involved
in their measurement of low enrichment of isotope
is high and so the numbers reported in their Table
1 do not have any positive or negative significance. The rate in which the isotope was infused
would give only a small enrichment (0.006 atom
% excess) of lSN in plasma alanine, which in turn
has to be obtained as a difference from two
relatively large values (0.371 and 0.365). After
three cycles of additions of reagents and dialysis
for removing free amino nitrogen and glutamine
amide nitrogen, alanine in the plasma (about 3
pmol) was enzymically degraded and the resulting
ammonia was aerated, absorbed, reduced to a
small volume and reacted with hypobromite
before freeze-trapping and being introduced into
the mass spectrometer. Even if it is granted that
no nitrogenous impurities were added during this
manipulation, and that miniaturized Rittenberg
tubes were used, contamination of an ultramicro-quantity of air will be a serious problem.
The observed very low or no enrichment of
nitrogen may partly be due to this method
adopted. Moreover, the 15N plasma enrichments are all reported after subtracting the preinfusion urinary ammonia nitrogen abundance.
When a difference of about 60 p.p.m. was
o 1981 The Biochemical Society and the Medical Research Society
350
M . Jeevanandam
expected, the corresponding plasma alanine/
glutamate I5N abundance from the pre-infusion
plasma sample should have been used since the
biological fractionations [7] are also of the same
order of magnitude.
Based on the results reported in their Table 1,
the authors have elaborated their discussion and
reasoned out to summarize that glycine cannot
be a ‘part of the cr-amino-nitrogen pool as
classically conceived’. The conclusion does not
agree even with their own other data. If the
enrichment of plasma glycine were about 25 times
that of urinary ammonia, then it would probably
mean that glycine nitrogen did not take part in
any transamination reactions and all the ammonia produced in the kidney was excreted.
However, they had seen that the enrichment of
plasma glycine was only seven times the urinary
ammonia enrichment. For glycine infusion one
would expect only this order of magnitude of
enrichment. If labelled alanine was administered
the ratio would be much smaller (1.85), showing
extensive exchange of nitrogen. Hence alanine
may be a better choice for rapid labelling of the
‘metabolic pool’ of nitrogen [Sl.
References
Ill JACKSON,A.A. & GOLDEN,M.H.N. (1980) I1sNI Glycine
metabolism in normal man: the metabolic d-amino nitrogen
pool. Clinical Sceince, 58,517-522.
121 SPRINSON,D.B. & RITTENBERG,
D. (1949) The rate of
utilization of ammonia for protein synthesis. Journal of
Biological Chemistry, 180,707-714.
131 SPRINSON,
D.B. & RITTENBERG,
D. (1949) The rate of
interaction of the amino acids of the diet with the tissue proteins.
Journalof Biological Chemistry, 180,715-726.
T. (1969) The measurement of
141 PIcOU, D.& TAYLOR-ROBERTS,
total protein synthesis and catabolism and nitrogen turnover in
infants in different nutritional states and receiving different
amounts of dietary protein. Clinical Science, 36,283-296.
[SI AQVIST,S.E.G. (195 I) Metabolic interrelationships among
amino acids studied with isotopic nitrogen. Acta Chemica
Scandinauica, 5 , 1046-1064.
161 LONG, C.L., JEEVANANDAM, M.,KIM,B.M. & KINNEY,J.M.
(1977) Whole body protein synthesis and catabolism in septic
man. AmericanJournalof ClinicalNufrition, 30,1340-1344.
171 GAEBLER,
O.H.,C H O ~H.C.,
,
V I ~ IT.G.
,
& VUKMIROVICH,
R. (1963) Significanceof I5N excess in nitrogenous compounds
of biological origin. Canadian Journal of Biochemistry and
Physiology, 41,1089-1097.
181 JEEVANANDAM,
M.,LONG, C.L. & KINNEY,J.M. (1979)
Kinetics of intravenously administered ”N-I-alanine in the
evaluation of protein turnover. American Journal of Clinical
Nutrition, 32,975-980.
AUTHORS’ REPLY
M.H.N. GOLDENAND A.A. JACKSON,
Tropical Metabolism Research Unit,
University of the West Indies, Mona, Kingston 7 ,Jamaica
Jeevanandam agrees that the concept of a single
metabolic pool of nitrogen is an oversimplification. To what extent does this oversimplification limit our understanding? There are at
least three different aspects of amino acid
metabolism; each may require a different
definition of the metabolic nitrogen pool. First,
amino acids comprise the precursor pool for
protein synthesis: this is quantitatively their
major role and is confined to a-amino nitrogen.
Secondly, individual amino acids have specific
metabolic pathways and functions, usually related
to non-amino metabolites. Thirdly, there are
specific pathways whereby nitrogen is transferred from molecule to molecule. All three
activities are taking place simultaneously and
each has an effect on the other. A full description
of nitrogen kinetics requires each to be defined.
The problems of definition are exemplified by
glutamine amide nitrogen, which should probably
be excluded from the metabolic nitrogen pool,
with respect to the precursor pool for protein
synthesis. However, there is no doubt that the
amide nitrogen plays an active and major role in
interorgan transport of nitrogen, and hence could
be considered part of the metabolic pool with
respect to this function. Similar considerations
have to be given to other non a-amino nitrogen.
Although Jeevanandam considers that it is well
known that all amino acids do not take part to the
same extent in nitrogen exchange, there are
difficulties in the interpretation of the data on
which this conclusion is based. In all the papers
we cited [ l ] large single unphysiological doses of
amino acids were given parenterally to experimental animals and the enrichment was measured
in the amino acids derived from mixed tissue
protein. Parenteral injection of large doses of
single amino acids themselves perturb metabolism. The ratio of enrichments in mixed protein
will depend on the amino acid profiles of the
individual proteins and their relative rates of
synthesis. There is not necessarily any equivalence between the ratio of the enrichment of
amino acids in mixed protein ‘and in the precursor free amino acid pool. After a single dose of
tracer, the enrichments in each compartment of
the free pool itself will change rapidly with time,