pathways of ammonia metabolism in the intact functioning...

Journal of Clinical InvestigationVol. 46, No. 7, 1967

Pathways of Ammonia Metabolism in the Intact FunctioningKidney of the Dog *

WILLIAM J. STONEt ANDROBERTF. PITTS t(From the Department of Physiology, Cornell University Medical College, New York, N. Y.)

Summary. Studies in which 15N-labeled precursors of urinary ammonia wereinfused into the artery of an intact functioning kidney of an acidotic dog haveled to the following conclusions: Preformed ammonia and ammonia derivedfrom the amide nitrogen of plasma glutamine are added directly to urinewithout significant incorporation into amino acid intermediates of renal tissue.Thus, reductive amination of a-ketoglutarate to form glutamate does not oc-cur to an appreciable extent nor is there significant transfer of the amide nitro-gen of glutamine to the corresponding keto acids to form glutamate, aspartate,alanine, or glycine. The enzyme system "glutaminase II" may participate toa significant extent in the metabolism of glutamine by forming aspartate andalanine by direct transamination of oxalacetate and pyruvate and liberatingthe amide nitrogen as ammonia. Renal alanine exists as a well mixed poolderived in roughly equal amounts from filtered and reabsorbed plasma alanineand newly synthesized alanine. The alanine pool of tubular cells does notequilibrate with the alanine of peritubular capillary blood. Transfer of thenitrogen of alanine to a-ketoglutarate and subsequent oxidative deaminationof the resulting glutamate can account for the ammonia formed from alanine.Glycine is not an important intermediate in renal nitrogen metabolism.

IntroductionIf an ammonia-generating enzyme cannot be

demonstrated in renal tissue in vitro [e.g.,L-amino acid oxidase (1), urease (2)], one mayreasonably infer that the reaction catalyzed bythat enzyme does not contribute to renal produc-tion of ammonia in vivo. However, the merepresence of an ammonia-generating enzyme in re-nal tissue does not necessarily indicate that it con-tributes significantly to the production of ammoniaby the intact functioning kidney. Certainly the ac-tivity of an enzyme under the highly artificial con-ditions of an in vitro assay cannot be related to itsfunctional contribution in vivo with any confidence.

* Submitted for publication January 23, 1967; acceptedMarch 15, 1967.

Aided by grants from the National Heart Institute ofthe National Institutes of Health (HE 00814, HTS 5264)and the Life Insurance Medical Research Fund.

tFellow of the New York Heart Association.t Address requests for reprints to Dr. Robert F. Pitts,

Dept. of Physiology,, Cornell University Medical College,1300 York Ave., New York, N. Y. 10021.

Enzymes known to be present in the kidney ofthe dog, and which are most likely to be involvedin ammonia production, include glutaminase I,glutamine transaminase plus an omega amidase(glutaminase II), a variety of L-amino acid trans-ferases, glycine oxidase (D-amino acid oxidase),and glutamate dehydrogenase. The latter is es-pecially interesting, because in vitro studies haveshown that the reaction which it catalyzes is re-versible and that equilibrium greatly favors thesynthesis of glutamate (3).

In the studies reported here, enzymatic path-ways of ammonia production by the kidney in vivowere studied by the infusion of a 15N-labeled pre-cursor into one renal artery of an acidotic dog.Both kidneys were removed, and the distributionof isotopic nitrogen among a limited number of thefree amino acids of kidney tissue and urinary am-monia was measured. The free tissue amino acidsstudied were limited to those which are stable andcan be isolated in pure form in an amount sufficientto permit accurate analysis of content of label,

1141

WILLIAM J. STONEAND ROBERTF. PITTS

namely glutamate, aspartate, glycine, and ala-nine. The number of precursors studied has beenlimited to those which we have synthesized in ourlaboratory, namely amide- and amino-labeled glu-tamines (4) and alanine (5). The number of ex-periments has been limited by the time involvedin the chromatographic separation and purifica-tion of the tissue amino acids before 15N analysis.

Wehave observed in the acidotic dog that theI5N of ammonium chloride and of amide-labeledglutamine infused into the renal artery appearsalmost exclusively in the urinary ammonia and isnot incorporated to any appreciable extent intothe free glutamate, aspartate, alanine, and glycineof kidney tissue. Thus, the glutamate dehydroge-nase system in vivo does not synthesize significantamounts of glutamate from a-ketoglutarate andammonia. Lacking labeled glutamate, 15N is notappreciably incorporated into other amino acidsby transamination. Furthermore, as one mightexpect, the course of the amide nitrogen of gluta-mine to urinary ammonia is direct and does notinvolve an amino acid intermediate.

In contrast, the 15N of amino-labeled glutamineappears in the urinary ammonia and in all of thefree amino acids of renal tissue that we have mea-sured. The extent of labeling of both aspartateand alanine relative to glutamate suggests that theglutaminase II system may be significantly in-volved in the metabolism of glutamine. Similarly,when the 15N is administered as alanine, the labelappears in the urinary ammonia and in all of thetissue amino acids studied. The distribution oflabel is consonant with the view that alanine ni-trogen is transferred to glutamate and subsequentlyappears as urinary ammonia and as tissue aspar-tate and glycine. In general, little label appearsin free tissue glycine no matter how the 15N isgiven.

Methods

Mongrel dogs of either sex weighing from 19 to 26 kgwere fed 10 g of ammonium chloride mixed with theirfood on each of the 3 days before an experiment. Afterintravenous injection of not more than 1 ml per kg of3% sodium pentobarbital, a constant infusion containing5 g creatinine, 22.5 g Na2SO4 10 H20, and 5 ml of 20op-aminohippurate (PAH) per L was administered in-travenously at 5 ml per minute. An endotracheal tubewas inserted to maintain the airway. A radio-opaquecatheter was passed into the left renal vein under fluoro-scopic visualization. A slow saline drip kept the catheter

patent. Both ureters were exposed through a midlinelower abdominal incision and catheterized to the levelof the renal pelvis. An indwelling needle with tightstylet was placed in the right femoral artery. The leftkidney was exposed retroperitoneally through a flankincision, and a curved 25-gauge needle connected to poly-ethylene tubing was introduced into the left renal arterynear its origin from the aorta.

The 1'N compound to be studied was dissolved in salineand infused into the left renal artery at a rate of ap-proximately 10lmoles per minute (0.97 ml per minute)throughout the course of the experiment. After a 20-minute equilibration period, urine was collected from eachkidney for 15 minutes. Over a period of 2 minutes at themidpoint of the clearance period, time-integrated sam-ples of arterial and renal venous blood were simultane-ously withdrawn into heparinized syringes. At the endof the period, the two kidneys were rapidly removed,weighed, and separately homogenized in 600 ml of satu-rated picric acid in paired Waring Blendors.

PAH was determined in whole blood and urine by themethod of Pilkington, Binder, De Haas, and Pitts (6).Renal blood flow (RBF) was calculated by the equationof Wolf (7), and renal plasma flow (RPF) was derivedfrom RBF and the measured arterial hematocrit. Cre-atinine in urine and plasma, ammonia in urine, arterialand renal venous plasma concentrations of amino acids,and "N content of urinary ammonia were analyzed bymethods previously described (4, 5, 8). Creatinine clear-ance was taken as a measure of glomerular filtration rate(GFR). Urinary pH was measured by a Radiometer-Copenhagen meter and a capillary glass electrode.

Kidney homogenates were centrifuged and filtered.Samples of 50 ml and 500 ml of each filtrate were sepa-rately treated according to the method of Tallan, Moore,and Stein (9). The smaller sample was flash evaporatedat 30° C and analyzed for acidic and neutral amino acidson a Beckman model 120 automatic analyzer. The largerwas similarly flash evaporated and used for chromato-graphic separation of glutamate, aspartate, alanine, andglycine. Glutamate and aspartate were removed andseparated by chromatographing the amino acid mixtureon Dowex 1 X 8 acetate, with 0.5 M acetic acid as theeluent. The eluate was collected in 5-ml fractions, spottedon filter paper, and sprayed with Ninhydrin to locatepeaks. Glutamine survives this treatment, and more than90% recoveries were obtained. Since no glutamate wasformed, preformed kidney glutamate is not diluted withglutamate formed on the Dowex 1 acetate column fromglutamine. The material emerging at column volume,including neutral and basic amino acids and ammonia,was flash evaporated, taken up in 25 ml of 0.1 N HCl,and heated for 30 minutes in a boiling water bath to hy-drolyze the glutamine. Although most of the glutamineis converted to pyrrolidone carboxylic acid, some glu-tamic acid is formed. This was removed by rechroma-tographing the residue on Dowex 1 acetate. The materialthat appeared at column volume was flash evaporated andchromatographed on Dowex 1 hydroxide, eluting with50 ml of distilled water to remove ammonia and basic

142

AMMONIAMETABOLISMIN THE KIDNEY

amino acids. The neutral amino acids were next elutedwith 0.5 M acetic acid, flash evaporated, and chromato-graphed on Dowex 50 X 4 (hydrogen form), eluting with1 N HCl (10). Fractions were spotted on filter paperbuffered with 4 M sodium acetate and sprayed withNinhydrin. Glycine and alanine peaks were identified,and the fractions corresponding to each were combinedand flash evaporated. At this stage all amino acid sam-

ples were analyzed individually for quantity and purityby automatic column chromatography. Glutamate andaspartate were usually obtained in pure form and inamounts adequate for 'N analysis without dilution withunlabeled material. Glycine and alanine were usuallyimpure, requiring repetition of chromatography one or

several times on the resin appropriate to the impurity.When ultimately purified, the amounts of alanine andglycine obtained (30 to 40 jsmoles) were insufficient for'N analysis. Therefore, 50 Amoles of the identical un-

labeled amino acid was added to each sample before con-

version to ammonia by treatment with Ninhydrin (11).Subsequent aeration and trapping of ammonia and con-

version to nitrogen gas for 15N analysis have been previ-ously described (4).

Results

Two experiments were performed with each offour "5N-labeled compounds: amide-labeled glu-tamine (94.9 atoms% 15N excess), amino-labeledglutamine (91.4 atoms% 15N excess), alanine(90.3 atoms% 15N excess), and ammonium chlo-ride (96.3 atoms% '-5N excess). Data obtainedin a typical experiment are presented in Table I.

Glomerular filtration rates, urine pH, weights,rates of ammonia excretion by the two kidneys,and concentrations of free amino acids in the tis-

sue of the two kidneys were comparable in alleight experiments.

Net extractions of plasma amino acids observedin the eight experiments are shown in Table II.

Since less than 1% of the filtered load of any

amino acid was excreted in the urine, the net ex-

traction of each amino acid, namely the arterialload in minus the venous load out, is essentiallyequal to rate of production or rate of utilization.Positive extraction is equated with utilization,negative extraction with production. The arterialload in is the product of arterial plasma concen-

tration and RPF. The total arterial load of theinfused amino acid, of course, is the sum of thearterial load, as calculated above, and the infusedload. The renal venous load out is the product ofthe renal venous plasma concentration and theRPF, corrected for both urine flow and rate ofintra-arterial infusion of fluid. Glycine and glu-tamine were consistently extracted from plasmain net amounts, whereas alanine and serine were

consistently produced and added to renal venous

blood. The small net extraction of threonine isof insignificant magnitude and, on the basis ofprevious studies, is considered negligible andwithin limits of experimental error.' These ob-servations are in agreement with those of Shal-houb and associates (8) and others (5, 12).

1 Plasma concentrations of aspartate are so low thatthey cannot be determined accurately. Production ofglutamate was low, as previously described (8). Hence,neither amino acid is included in Table II.

TABLE I

Data obtained in a representative experiment in which amide-15N-kabeled glutamine wasinfused into the left renal artery of an acidotic dog*

Elapsedtime

Ammonia KidneyGFR Urine flow Urine pH excretion Urine ammonia-15N weight

RPFL R L L R L R L R L R L-R L R

min ml/min jsmoles/min atoms %excess g0 Begin infusion of amide-55N glutamine (94.9 atoms %axcess) into left renel artery at 9.92 Mumoles/min

20-35 45.2 49.9 199 2.43 3.09 5.63 5.73 43.4 44.1 5.662 1.056 4.606 67.9 67.535-37 Remove kidneys and homogenize in picric acid.

Amino acid Plasma concentration Kidney concentration

Arterial Renal venous L Rumotes/ml jtmotes/g wet wt

Threonine 0.097 0.102 0.141 0.154Serine 0.117 0.161 0.277 0.285Glutamine 0.452 0.304 0.390 0.362Glycine 0.169 0.149 0.594 0.623Alanine 0.365 0.467 0.892 0.896Glutamate 0.015 0.020 4.04 4.15Aspartate 0.008 0.008 1.03 0.992

* Abbreviations: GFR= glomerular filtration rate, RPF = renal plasma flow, L = left, R = right.

1143


TABLE II

Summary of arteriovenous extractions of representative amino acids by the left kidney duringthe infusion of labeled compounds into the left renal artery

Net extraction from plasmaExperi-ment Compound infused Threonine Serine Glutamine Glycine Alanine

jhmoles/min1 Amino-15N glutamine -0.4 - 5.7 +29.1 +3.0 - 5.42 -0.7 - 6.0 +20.7 +4.1 - 9.3

3 Ammonium chloride-15N -0.8 - 7.1 +21.5 +4.7 - 4.54 -0.9 - 6.2 +24.0 +3.0 -12.6

5 Amide-15N glutamine -0.9 - 8.5 +39.9 +4.2 -19.76 +0.5 - 5.1 +28.9 +3.8 -10.9

7 Amino-15N alanine -0.2 - 8.8 +42.4 +4.9 -13.88 -0.4 -10.2 +34.9 +6.2 -13.1

Mean -0.5 - 7.2 +30.2 +4.2 -11.2

Table III summarizes the specific activities(atoms per cent excess 15N) of left and right uri-nary ammonias and of the free tissue amino acidsof left and right kidneys. Subtraction of the spe-cific activity of the urinary ammonia or of a tis-sue amino acid of the noninfused kidney from thecorresponding specific activity of urine or tissueof the infused kidney corrects for recirculation oflabel in any form. Corrected plasma specific ac-tivity is calculated by dividing the known rate ofinfusion of 15N by the total arterial load of thatamino acid (perfused load + infused load, as de-scribed previously). Division of the specific ac-tivity of the compound measured in urine or in kid-ney (corrected for recirculation) by the plasmaspecific activity of the compound infused (similarlycorrected for recirculation) gives the per cent ofthe product derived from the precursor.

Figure 1 demonstrates that ammonia nitrogen,presented to the kidney as preformed ammonia inthe blood (A) or produced in the kidney from theamide nitrogen of plasma glutamine (B), is notsignificantly incorporated into the free aspartate,glutamate, glycine, or alanine of renal tissue.Thus, in the kidney of the acidotic dog, glutamatedehydrogenase does not catalyze the incorporationof significant amounts of labeled ammonia intoglutamate.

It must be emphasized that the significance ofthe per cent of the urinary ammonia derived fromblood ammonia is basically different from that ofthe per cent of the urinary ammonia derived fromeither nitrogen of plasma glutamine. Normallythe concentration of arterial ammonia is very

low (13-15). In our experiments the quanti-ties of 15NH4Cl infused are by no means traceramounts relative to the normal arterial load ofammonia. Accordingly, the per cent of the uri-nary ammonia derived from the blood ammoniawill, within limits, vary as a direct function of therate of infusion of 15NH4Cl. The same is not trueof experiments in which "5N-labeled amino acidsare infused and in which the infused load of the'5N-labeled amino acid can be considered as atracer quantity relative to the much larger normalperfused load of unlabeled amino acid.

Incorporation of label into amino acids is strik-ingly different when 15N is infused as amino-la-beled glutamine or alanine, as shown by the ex-periments summarized in Figure 2, A and B.Significant amounts of '5N are now incorporatedinto all of the free tissue amino acids studied.Presumably, such incorporation occurs by reac-tions involving transamination, certainly not byreductive amination of a-ketoglutarate to formglutamate (see Figure 1, A and B).

Differences in per cent of ammonia, glutamate,aspartate, and so forth derived from a plasma pre-cursor are related to each other in the same pro-portions as are the differences in the extent oftheir 15N labeling. This derives from the fact thatthe percentages, in any one experiment, are atomsper cent excess 15N of the several products factoredby a commonatoms per cent excess '5N of the pre-cursor, times 100. It is evident from Figure 2, A,that the labeling of aspartate is greater than thatof glutamate, an observation which indicates thatthe aspartate could not have been derived solely

1144


TABLE III

Incorporation of 15N into urinary ammonia and into representative free amino acids of renal tissueduring the infusion of labeled compounds into the left renal artery

Specific activity %DerivedExperi- from infused

ment Compound analyzed L R L-R compound

atoms %ISN excess

Amino-15N glutamine (SA arterial plasma: exp. 1 = 14.6, exp. 2 = 14.7 atoms %excess)1 Urinary ammonia 2.758 0.799 1.9592 2.623 1.025 1.598

1 Kidney aspartate 6.776 1.523 5.2532 6.585 1.871 4.714

1 Kidney glutamate 4.985 1.074 3.9112 4.370 1.370 3.000

1 Kidney alanine 4.614 1.365 3.2492 4.540 1.637 2.903

1 Kidney glycine 0.600 0.399 0.2012 0.986 0.409 0.577

Ammonium chloride-15N (SA arterial plasma: exp. 3 = 35.7, exp. 4 = 28.7 atoms %excess)Urinary ammonia

Kidney aspartate

Kidney glutamate

Kidney alanine

Kidney glycine

Amide-15N glutamine (SA arterial plasma: exp. 5

5 Urinary ammonia6

5 Kidney aspartate6

5 Kidney glutamate6

5 Kidney alanine6

5 Kidney glycine6

17.4814.92

0.3640.389

0.3480.296

0.2310.270

0.0150.060

= 9.43, exp. 6 =

5.6625.090

0.0870.152

0.0830.091

0.1220.105

0.1190.029

1.6390.940

0.0620.065

0.0560.124

0.0480.093

0.0040.043

8.80 atoms %

1.0561.151

0.0370.054

0.0750.041

0.0340.056

0.0250.021

15.8413.98

0.3020.324

0.2920.172

0.1830.177

0.0110.017

excess)

4.6063.939

0.0500.098

0.0080.050

0.0880.049

0.0940.008

Amino-16,N alanine (SA arterial plasma: exp. 7 = 6.39; exp. 8 = 6.27 atoms %excess)

7 Urinary ammonia 0.504 0.197 0.3078 0.503 0.167 0.336

7 Kidney aspartate 1.036 0.481 0.5558 1.646 0.413 1.233

7 Kidney glutamate 0.868 0.288 0.5808 1.182 0.292 0.890

7 Kidney alanine 3.853 1.054 2.7998 4.766 0.970 3.796

7 Kidney glycine 0.230 0.099 0.1318 0.294 0.107 0.187

13.410.9

36.032.1

26.820.4

22.319.8

1.383.93

44.448.8

0.851.13

0.820.60

0.510.62

0.030.06

48.944.8

0.531.11

0.090.57

0.930.56

1.000.09

4.805.36

8.6919.7

9.0814.2

43.860.6

2.052.98

34

34

34

34

34

1145


15N AMMONIUMCHLORDEINFUSED

A. 70r

15N AMIDE GLUTAMINE INFUSED

B.60F

/

EE 50-c,

X0 , 40*C *o 0

0 0z 20

E I0,41

Urine Kidney Kidney KidneyAmmonia. Glutamate Aspartate Alanine

KidneyGlyclne

I.

F

FE

1*

///

//

AUrine Kidney Kidney Kidney Kidney

Amrnonia Glutamate Asportato Alanine Glycine

FIG. 1. PERCENTAGESOF URINARY AMMONIAAND OF FREE ALANINE, ASPARTATE, GLUTAMATEANDGLYCINE OF KIDNEY

TISSUE DERIVED FROMCIRCULATING PLASMAPRECURSORS. A, percentages derived from blood ammonia. B, percentagesderived from the amide nitrogen of glutamine. A and B, each the mean of two experiments; ranges indicated bydashed lines.

from a uniformly labeled pool of glutamate. Thus,aspartate must be derived, at least in part, froma more highly labeled precursor.

It is apparent from Figure 2, B, that the alanineof kidney tissue becomes highly labeled when15N alanine is infused into the renal artery. Amean of some 52%o of kidney alanine had its ori-gin from plasma alanine in these experiments;

15N AMINO GLUTAMINEINFUSED

48%o was derived from alanine synthesized de novofrom unlabeled precursors by tubular cells.

It is also evident from Figure 2, B, that gluta-mate could readily acquire its label from alanineby transamination of a-ketoglutarate. Perhapseven the aspartate, which has a slightly higher la-bel than glutamate, might be derived by transami-nation of oxalacetate by glutamate if one discounts

15N ALANINE INFUSED

A.60[

ii.

ii.

/I.

Urine Kidney Kidney KidneyAmmonia Glutamate Aspartate Alanine

70r

60j

2 500

h.

_ a 30

, 20

Il0

KidneyGlycine

1[I.t

/

Urine Kidney Kidney KidneyAmmonia Glutomote Asportte Alanine

FIG. 2. PERCENTAGESOF URINARY AMMONIAAND OF FREE ALANINE, ASPARTATE, GLUTAMATE, AND GLY-CINE OF KIDNEY TISSUE DERIVED FROMCIRCULATING PLASMA PRECURSORS. A, percentages derived fromamino nitrogen of glutamine. B, percentages derived from amino nitrogen of alanine. A and B, eachthe mean of two experiments; ranges indicated by dashed lines.

70r

60w-

20._

E

~02._ E

0 X0.0- .00

m

50 F40F

301

201

l0

TOr

C

2E 5C'

0

n 40

zO0D a 30c

I oZ20

B.

KidneyGlycine

1146

--_-

,,

7--


the small difference. In any event, the ammoniais probably derived from glutamate by oxidativedeamination. Clearly, neither aspartate nor glu-tamate could acquire its label from tissue am-monia by reductive amination.

Discussion

Calculation of the per cent of a renal productderived from a precursor delivered to the kidneyin arterial blood requires that a steady state exist.Thus, the specific activity of a precursor in renaltissue relative to its specific activity in plasma mustbe constant and independent of time. In all of ourexperiments, the labeled compound was infusedinto the renal artery at a constant rate for 20 min-utes before the start of the clearance period. Weinferred that a steady state was achieved duringthis time with respect to uptake and turnover oflabeled precursor and formation of labeled product.Steady state conditions very probably existed inexperiments in which glutamine was infused anddemonstrably existed in experiments in whichalanine and ammonia were infused.

Pitts, Pilkington, and De Haas (4) observedthat the time for attainment of constancy of la-beling of urinary ammonia after starting an intra-arterial infusion of amide-labeled glutamine is lessthan 5 minutes. This fact, coupled with the smallintrarenal pool of glutamine and high rate of ex-traction of glutamine from plasma (8), indicatesthat the glutamine pool of the kidney turns overrapidly. Since essentially no glutamine is syn-thesized by the dog kidney (16), it follows thatthe atoms per cent excess 15N of renal glutaminemust become equal to the atoms per cent excess15N of plasma glutamine within less than 5 min-utes. The fact that we have been unable to isolateglutamine from renal tissue in pure form inamounts sufficient for accurate 15N analysis hasprevented a direct test of this thesis.

Such a test is possible for alanine, and its basisis illustrated in Figure 3. The situation is com-plicated by the fact that alanine is synthesizedwithin the kidney in amounts approximating thosefiltered and reabsorbed. In the experiment sum-marized in Figure 3, the perfused load of alaninewas 93.8 ptmoles per minute (product of plasmaalanine concentration and RPF). Seven Mmolesper minute of alanine containing 6.32 pmoles 15Nexcess was infused into the renal artery. Cor-

Ftrfused Infused Total RbstglomerularLoad Load Load Load

93.8 7.0 79.5

21.3

2.14

Synthesized15.8

Reabsorbed A21.3-+37.1 0

2.7f~Cnete'd t~o

NH3

79.534.4113.9

0.56

FIG. 3. RENAL BALANCE OF ALANINE, ILLUSTRATING

THE CONTRIBUTIONSOF FILTERED AND REABSORBEDALANINE

TO THE TOTAL RENALPOOL.

rected specific activity of the total arterial loadwas thus 6.32/100.8 x 100 = 6.27 atoms %o excess.The filtered load of alanine was 21.3 umoles perminute (product of GFRand plasma alanine con-centration). Obviously, the filtered alanine musthave had the same specific activity as the plasmaalanine. Since all was reabsorbed, 21.3 jumoles ofalanine, which had the same specific activity asplasma, entered tubular cells from the lumeneach minute. Reabsorbed alanine-15N thereforeamounted to 1.336 pmoles excess (21.3 jimoles X6.27 atoms % excess/100). Alanine was synthe-sized 2 from unlabeled nitrogen sources and addedto the renal pool at a rate of 15.8 1&moles per min-ute. The pool turned over at a rate of 37.1 1imolesper minute (21.3 + 15.8). Dividing 15N alanineadded to the renal pool each minute (1.336Mmoles) by rate of turnover of the pool (37.1Mmoles) and multiplying by 100 gives an apparentspecific activity of renal alanine of 3.60 atoms, %'5N excess. The measured specific activity ofrenal alanine was 3.796 atoms % 15N excess, a

2 The rate of synthesis of alanine is a calculated mini-mal one. It is derived from the measured net positiveextraction of alanine (113.9 - 100.8 = 13.1 umoles perminute) plus the measured conversion of alanine to uri-nary ammonia (2.14 Amoles per minute) plus the esti-mated contribution of alanine to the ammonia added torenal venous blood (0.56 /Amoles per minute).

1147


value 105%o of the predicted one. Therefore, after20 minutes of intra-arterial infusion a steady statehas been attained, such that the 15N of the alanineof the renal pool has become a constant fraction ofthe 15N of the alanine of arterial plasma. Thesecalculations also indicate that alanine enters tubu-lar cells only through their luminal surfaces as a

consequence of filtration and reabsorption. Equili-bration of tissue alanine with alanine of peritubu-lar capillary blood does not occur to any appre-

ciable extent.Renal ammonia, like renal alanine, is a mixture

of that taken up by tubular cells from blood andthat synthesized within tubular cells from a varietyof precursors. Our evidence (17) indicates that15NH4Cl infused into a renal artery distributesthroughout a well-mixed pool, which includes notonly tubular cells but also tubular urine and peri-tubular blood, all phases having a common specificactivity. This provides substance for the ex-

pressed view that the free base is in diffusionequilibrium among all phases of the renal cortex(14, 18).

In Figures 1 and 2, percentages of urinary am-

monia derived from given precursors are com-

pared with percentages of tissue amino acids de-rived from those same precursors. For the rea-

sons stated above one could equally well desig-nate the bar marked urinary ammonia as tissueammonia.

Figure 1 demonstrates that, in the acidotic dog,the glutamate dehydrogenase reaction, glutamate+ NAD+ = a-ketoglutarate + NH8 + NADH+H although reversible, does not proceed appre-

ciably in the direction of glutamate synthesis. Theproduction of ammonia and a-ketoglutarate fromglutamate presumably is favored by a high ratio ofNAD+ to NADH, the removal in urine or bloodof the ammonia formed in the reaction, a highconcentration of tissue glutamate relative toa-ketoglutarate, or unknown factors. The abovefindings do not mean that the acidotic kidney doesnot form glutamate from a-ketoglutarate, only thatthe glutamate dehydrogenase pathway is of minorsignificance. Our evidence (19) shows that thelabel of a-ketoglutarate-5-14C is rapidly incorpo-rated into renal glutamate, presumably throughtransamination by a number of amino acids, whichultimately yield their amino nitrogen as ammonia(20, 21).

Figure 2, A, demonstrates that the 15N of amino-labeled glutamine appears in the urinary ammoniaand in tissue glutamate, aspartate, alanine, andglycine. The extent of the labeling of tissue as-partate very significantly exceeds that of tissueglutamate. Therefore, the aspartate pool of renaltissue could not have acquired its label from a com-mon pool of glutamate labeled only to the degreeshown in Figure 2, A. It must have acquired its15N from a precursor more highly labeled thanthis common pool. Two possibilities are evident.1) The aspartate could acquire its label, at leastin part, from plasma and tissue glutamine bydirect transamination of oxalacetate by the glu-taminase II reaction. Glutamine + oxalacetate -*

aspartate + a-ketoglutaramate; a-ketoglutaramate-> a-ketoglutarate + NH3. 2) The aspartate couldacquire its label as a consequence of conventionaltransamination of oxalacetate by a compartmentedhighly labeled pool of glutamate. Oxalacetate +glutamate = aspartate + a-ketoglutarate. Thehighly labeled glutamate could represent a smallintramitochondrial pool of glutamate formed fromglutamine by the glutaminase 1 reaction and notyet transferred to the lesser labeled cytoplasmicpool.

From Figure 2, A, it would appear that tissuealanine could have acquired its label from the com-mon pool of glutamate by transamination of py-ruvate. Actually it could not, for the renal poolof alanine is a mixture of unlabeled, filtered, andreabsorbed alanine plus newly synthesized ala-nine, which derived its label ultimately from theamino nitrogen of tissue glutamine. Highly la-beled synthesized alanine plus unlabeled, filtered,and reabsorbed alanine constitute the mixture ana-lyzed as tissue alanine. From the calculationspresented in the first part of the discussion, it isevident that the newly synthesized alanine musthave had a specific activity more than twice thatof the mixed renal pool.

Wehave no data that establish either of the twoexplanations cited above for the high labeling ofalanine and aspartate relative to glutamate. Inrat liver, glutaminase I is a mitochondrial enzyme(22). Therefore, glutamate formed by the deami-nation of glutamine must be compartmented tosome extent within mitochondria. However, thebulk of the glutamate-pyruvate transaminase -ofrat liver is extramitochondrial in locus (23).

148


Hence, highly labeled glutamate might have toenter the less labeled cytoplasmic pool to trans-aminate pyruvate effectively. The alanine labelwould approach that of the common pool of glu-tamate. In contrast, the pyruvate-activated glu-taminase II of rat liver is entirely cytoplasmic(22). Direct transamination of pyruvate by cyto-plasmic glutamine could form highly labeled ala-nine. This argument is not conclusive, for theintracellular loci of enzymes in the dog kidney arenot known and may differ from those of rat liver.Furthermore, in rat liver the bulk of the glutamate-oxalacetate transaminase is contained within mito-chondria (24). Additionally, studies on partiallypurified glutamine transaminase of rat liver havedemonstrated only minor direct transfer of theamino nitrogen of glutamine to oxalacetate (25).No studies on transfer activities of the enzyme ofdog kidney are available, but they may differ fromthose of rat liver. Richterich and Goldstein (26)and Pollak, Mattenheimer, DeBruin, and Wein-man (27) have observed that the kidney of thedog contains active glutamine transaminase andglutaminase. Our data suggests that glutaminetransaminase may play a significant role in the syn-thesis of ammonia, aspartate, and alanine but donot prove it.

Figure 2, B, shows that 52%, of kidney alanineis derived from plasma alanine and 48% is syn-thesized within the kidney. This latter moietyacquires its nitrogen in large part from the aminogroup of glutamine. Obviously, in these experi-ments, glutamate could derive its label by trans-amination of a-ketoglutarate by tissue alanine.Labeled glutamate could, in turn, account for thelabel that appears in urinary ammonia by way ofthe glutamate dehydrogenase pathway. Aspartateand glutamate are labeled to approximately thesame extent. Hence, aspartate might be formedfrom oxalacetate by transamination with gluta-mate, the commonly inferred final step in the se-quence of alanine to glutamate to aspartate. If,however, the labeling of aspartate significantly ex-ceeds that of glutamate, a more direct transferfrom a more highly labeled source would be indi-cated, as discussed above.

Glycine is not labeled to an appreciable extentby any of the 15N-labeled compounds infused, afact which supports the observation that glycine ofplasma plays only a minor role in renal ammonia

formation (4). Thus, glycine appears to beneither a major plasma precursor nor a majortissue intermediate of the ammonia produced bythe kidney.

References1. Blanchard, M., D. E. Green, V. Nocito, and S. Ratner.

L-Amino acid oxidase of animal tissue. J. biol.Chem. 1944, 155, 421.

2. Archibald, R. M. Chemical characteristics and physi-ological roles of glutamine. Chem. Rev. 1945, 37,161.

3. White, A., P. Handler, and E. L. Smith. Principlesof Biochemistry, 3rd ed. New York, McGraw-Hill, 1964, p. 488.

4. Pitts, R. F., L. A. Pilkington, and J. C. M. De Haas.1N tracer studies on the origin of urinary am-monia in the acidotic dog, with notes on the en-zymatic synthesis of labeled glutamic acid and glu-tamines. J. clin. Invest. 1965, 44, 731.

5. Pitts, R. F., and W. J. Stone. Renal metabolism ofalanine. J. clin. Invest. 1967, 46, 530.

6. Pilkington, L. A., R. Binder, J. C. M. De Haas, andR. F. Pitts. Intrarenal distribution of blood flow.Amer. J. Physiol. 1965, 208, 1107.

7. Wolf, A. V. Total renal blood flow at any urine flowor extraction fraction (abstract). Amer. J. Phys-iol. 1941, 133, 496.

8. Shalhoub, R., W. Webber, S. Glabman, M. Canessa-Fischer, J. Klein, J. DeHaas, and R. F. Pitts.Extraction of amino acids from and their additionto renal blood plasma. Amer. J. Physiol. 1963,204, 181.

9. Tallan, H. H., S. Moore, and W. H. Stein. Studieson the free amino acids and related compounds inthe tissues of the cat. J. biol. Chem. 1954, 211, 927.

10. Hirs, C. H. W., S. Moore, and W. H. Stein. Thechromatography of amino acids on ion exchangeresins. Use of volatile acids for elution. J. Amer.chem. Soc. 1954, 76, 6063.

11. Wilson, D. W., A. 0. C. Nier, and S. P. Reimann.Preparation and Measurement of Isotopic Tracers.Ann Arbor, J. W. Edwards, 1964, p. 34.

12. Owen, E. E., and R. R. Robinson. Amino acid ex-traction and ammonia metabolism by the humankidney during the prolonged administration of am-monium chloride. J. clin. Invest. 1963, 42, 263.

13. Nash, T. P., Jr., and S. R. Benedict. The am-monia content of the blood, and its bearing on themechanism of acid neutralization in the animalorganism. J. biol. Chem. 1921, 48, 463.

14. Denis, G., H. Preuss, and R. Pitts. The pNHs ofrenal tubular cells. J. clin. Invest. 1964, 43, 571.

15. Pilkington, L. A., J. Welch, and R. F. Pitts. Re-lationships of pNHs of tubular cells to renal pro-duction of ammonia. Amer. J. Physiol. 1965, 208,1100.

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16. Krebs, H. A. Metabolism of amino-acids. IV. Thesynthesis of glutamine from glutamic acid and am-monia, and the enzymic hydrolysis of glutamine inanimal tissues. Biochem. J. 1935, 29, 1951.

17. Stone, W. J., S. Balagura, and R. F. Pitts. Unpub-lished data.

18. Pitts, R. F. Renal production and excretion of am-monia. Amer. J. Med. 1964, 36, 720.

19. Lyon, M. L., and R. F. Pitts. Unpublished data.20. Lotspeich, W. D., and R. F. Pitts. The role of

amino acids in the renal tubular secretion of am-monia. J. biol. Chem. 1947, 168, 611.

21. Kamin, H., and P. Handler. The metabolism ofparenterally administered amino acids. III. Am-monia formation. J. biol. Chem. 1951, 193, 873.

22. De Duve, C., R. Wattiaux, and P. Baudhuin. Distri-bution of enzymes between subcellular fractions inanimal tissues. Advanc. Enzymol. 1962, 24, 291.

23. Kafer, E., and J. K. Pollak. Amino acid metabolismof growing tissues. II. Alanine-glutamic acidtransaminase activity of embryonic rat liver.Exp. Cell Res. 1961, 22, 120.

24. Eichel, H. J., and J. Bukovsky. Intracellular distri-bution pattern of rat liver glutamic-oxalacetictransaminase. Nature (Lond.) 1961, 191, 243.

25. Meister, A., and S. V. Tice. Transamination fromglutamine to a-keto acids. J. biol. Chem. 1950,187, 173.

26. Richterich, R. W., and L. Goldstein. Distributionof glutamine metabolizing enzymes and productionof urinary ammonia in the mammalian kidney.Amer. J. Physiol. 1958, 195, 316.

27. Pollak, V. E., H. Mattenheimer, H. DeBruin, andK. J. Weinman. Experimental metabolic acido-sis: the enzymatic basis of ammonia productionby the dog kidney. J. clin. Invest. 1965, 44, 169.

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