UREA METABOLISM IN MAN Mackenzie Walser, Leonard J. Bodenlos J Clin Invest. 1959;38(9):1617-1626. https://doi.org/10.1172/JCI103940. Research Article Find the latest version: https://jci.me/103940/pdf UREA METABOLISM IN MAN * By MACKENZIE WALSERt AND LEONARD J. BODENLOSt (From the Naval Medical Research Institute, National Naval Medical Center, Bethesda, Md. and the Laboratory of Kidney and Electrolyte Metabolism, National Heart Institute, National Institutes of Health, Bethesda, Md.) (Submitted for publication March 26, 1959; accepted May 14, 1959) Urea has long been considered to be meta- C"4-labeled urea containing 1 mc. of C14 per mMole was obtained from Volk Laboratories under authorization by bolically inert. However, recent studies with the Atomic Energy Commission. Both preparations were isotopic urea in several species of animals have prepared for use by dissolving in sterile saline solution. revealed that urea is hydrolyzed in the alimen- Tests for pyrogenicity and bacterial contamination were tary tract even when administered parenterally negative. (1-5). This breakdown is presumably due to The subjects were normal healthy volunteers who con- tinued their usual meals and activities during the studies. the action of bacterial urease, since urea metab- One glass of water per hour was ingested for four to eight olism can be eliminated or reduced to negligible hours following administration of labeled urea. Urinary proportions by the administration of antibiotics. catheters were not employed. In the studies reported herein, labeled urea was Measured portions of labeled urea (25 to 100 mg. of administered intravenously to normal subjects N" -urea or 2.5 Mc. of C"4-urea) were injected intravenously. At intervals, venous blood samples were obtained, and and the rate of extrarenal disposal was deter- immediately thereafter, the subjects voided. In the N'5 mined. Both N15- and C14-labeled materials were experiments, the urine samples were acidified immediately employed in order to minimize the problem of to prevent loss of ammonia. reincorporation of tracer atoms into urea. In The nitrogen content and atoms per cent excess N1" were the case of N'5, enriched ammonia produced by determined in urinary ammonia, urea and total nitrogen and in serum urea. Urine ammonia was isolated on per- urea hydrolysis in the gut becomes reincorpo- mutit which was then washed five times with distilled rated into newly formed urea. In the case of water. Ammonia-free urine and a tungstic acid filtrate of C14, recent evidence (6) suggests that reincorpo- serum were treated with urease according to the method ration into urea of labeled CO2, formed by bac- of Van Slyke and Cullen (8). A sample of urine was also terial ureolysis in the gut, may also occur; a subjected to Kjeldahl digestion overnight. After the addi- the tion of alkali, ammonia from these various fractions was conclusion supported by present experiments. steam distilled into boric acid solution and titrated with Despite these limitations, the results indicate 0.1 N HCl. The recovery of urea was determined simul- that 15 to 30 per cent of the urea being synthe- taneously in each experiment and averaged 98 per cent. sized in normal subjects is continually being The solutions remaining after titration were evaporated to destroyed. This breakdown can be eliminated a small volume and allowed to react with sodium hypo- by oral administration of neomycin. bromite in Rittenberg tubes. The atoms per cent N" in the evolved nitrogen gas was determined in the mass spec- trometer (Consolidated model 21-201). Atoms per cent METHODS excess N"6 was determined by subtracting values for atoms Urea containing 60.9 atoms per cent N"5 was synthesized per cent N"6 in control samples of blood and urine. Com- by the ammonolysis of diphenyl carbonate (7) and purified pared to an ammonium chloride standard, the control by recrystallization.' The resulting product had a melting samples contained a biological excess of N"6 of less than point of 130° C. (uncorrected). The nitrogen content was 0.004 atoms per cent. The maximum error of the N"6 46.7 per cent (calculated 46.7 per cent). analyses was a0.002 atoms per cent. In the C" experiments, total urea was determined by * An abstract of this work has been published (Urea the method of Conway (9). C"4-urea was determined as breakdown in normal man. J. clin. Invest. 1957, 36, 933). follows: aliquots of blood or urine were acidified with HCl The opinions presented herein are those of the authors and and shaken with a few drops of caprylic alcohol in order to do not necessarily reflect the views of the United States remove all bicarbonate. Sufficient alkali was added to Navy. restore the pH to approximately 7, and aliquots were placed t Formerly Lieutenant, Medical Corps, USNR. Pres- in 50 ml. flasks containing a center well. One ml. of 3 ent address: Johns Hopkins School of Medicine, Balti- per cent urease solution (Sigma Chemical Co.) and 1 ml. more, Md. of phosphate buffer, pH 7.2, were added and the flasks Lieutenant Commander, Medical Service Corps, USN. were stoppered. After 20 minutes shaking at 370 C., dilute Dr. W. J. McCarville kindly carried out this synthesis. H2SO4 was injected through the stopper. Shaking was 1617 1618 MACKENZIE WALSER AND LEONARD J. BODENLOS continued for another hour. In the earlier experiments, designate the difference between the extrapolated intercept the center well contained 25 per cent NaOH, C02-free, value, M., and the amount injected, M (which is the inter- which was transferred to centrifuge tubes, mixed with 10 cept value which would have obtained if equilibration had ml. 1 per cent BaCl2 in C02-free water, and centrifuged. occurred instantaneously): The precipitated barium carbonate was washed with C02- Mo= M - x. free 50 per cent ethanol, suspended in 95 per cent ethanol 4) and transferred to weighed planchets. The planchets were Substituting in Equation 3, we have, for any time greater dried, reweighed and counted in a flow counter for a total than the equilibration period: of 10,000 counts. After correction for background and Mt = (M - x) e-(A vP)t. 5) self-absorption, the results were expressed in terms of an The apparent volume of aliquot of the injected solution analyzed simultaneously. distribution, V, is given by: In M0 M-x the later experiments, the center well contained 0.2 VP= P 6) ml. of Hyamine®, 0.5 N. This was transferred with the PO Po aid of 1.5 ml. toluene to specimen vials, mixed with 15 ml. where po is the intercept value of the plasma concentration toluene containing diphenyl oxazole, 1 N, capped, cooled of tracer. The plasma concentration of tracer at time t, and counted in a Tri-Carb liquid scintillation counter for pt (where t is greater than the equilibration period), is a total of 5,000 counts. The results were expressed in terms of an aliquot of the injected solution analyzed pt = po e-(AIVP)t. 7) simultaneously. Therefore, po and A/VP may be derived from observa- tions of pt. In order to calculate V it is necessary to have CALCULATIONS an estimate of x, as Equation 6 shows. If the rate of excretion of native substance, U, is large When a trace quantity, M, of a substance normally with respect to the rate of metabolism, F, then a good present in the blood is injected intravenously, the tracer estimate of x can be obtained by observing the excretion molecules become mixed with the native molecules present of tracer, as shown in the following derivation. in the blood and in the interstitial fluid. Under certain The rate of renal (or renal plus extrarenal) excretion of conditions the tracer molecules eventually become evenly tracer, ut, is determined by the fact that the specific mixed with all of the native substance present in the body activities (or, in the case of stable isotopes, the atoms per in an exchangeable form. Although this completely uni- cent excess) in blood and urine are equal with the qualifi- form ratio of tracer to native molecules is theoretically cations noted below: in a closed for unobtainable except system, practical pur- Ut _ Pt poses an equilibrium distribution may be obtained because U P, 8) the rates of excretion and/or metabolic destruction are slow relative to the rate of equilibration. Substituting for pt from Equation 7, If the rates of formation, A, excretion, U, and metabolic destruction, F, of the native substance remain constant ut = -F;poPo e(A/VP)t 9) during the period of observation, and if The total amount of tracer, S, excreted up to time t (pro- A = U +F, 1) vided that t is greater than the equilibration period) is then the plasma concentration, P, of native substance will therefore: remain constant, as will VP, the total amount in the body, where V is its apparent volume of distribution (assuming S = X + A = X+ lUVPEl -e-(A/VP)t]. 10) that V does not change). Following equilibration of injected tracer, the amount, Dividing by M, the amount of tracer injected, we have of tracer at will decline S x + UVp [1 Mt, remaining any given time, t, - e-(A/VP)t]. 11) at a rate given by the turnover rate of native substance, MM AM A/VP. It is assumed here that no tracer returns to the This equation can be treated as a straight line function of pool once metabolized: two variables, viz., S/M and [1 -e-(A/VP)tJ, following the suggestion of Berkson, Keating, Power and McConahey - dMt/dt = V Mt. 2) (10).