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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,