Renal ammoniagenesis in an early stage of
metabolic acidosis in man.
A Tizianello, … , N Acquarone, G M Ghiggeri
J Clin Invest.
Total renal ammonia production and ammonia precursor utilization were evaluated in
patients under normal acid-base balance and in patients with 24-h NH4Cl acidosis by
measuring (a) ammonia excreted with urine and that added to renal venous blood, and (b)
amino acid exchange across the kidney. In 24-h acidosis not only urinary ammonia
excretion is increased, but also total ammonia production is augmented (P less than 0.005)
in comparison with controls. By evaluating the individual role of acid-base parameters, urine
pH and urine flow in influencing renal ammonia production, it was shown that the degree of
acidosis and urine flow are likely major factors stimulating ammoniagenesis. Both urine pH
and urine flow are determinant in the preferential shift of ammonia into urine. In 1-d acidosis,
renal extraction of glutamine was not increased and the total ammonia produced/glutamine
N extracted ratio was higher than in controls (P less than 0.005) and was inversely
correlated with the log of arterial bicarbonate concentration (P less than 0.001). In the same
condition, renal glycine and ornithine uptake took place; the more severe the acidosis, the
greater was the renal extraction of these amino acids (P less than 0.001). These data
indicate that at the early stages of metabolic acidosis, in spite of a brisk increase in
ammonia production, the mechanisms responsible for the increased glutamine […]
ALBERTO TIZIANELLO, GIACOMO DEFERRARI, GIACOMO GARIBOTTO,
CRISTINA ROBAUDO, NICOLA ACQUARONE, and GIAN MARCO GHIGGERI, Istituto
Scientifico di MedicinaInterna, Servizio
ofGenoa, 16132, Genoa, Italy
R A C T
ammonia production and
ammonia precursor utilization were evaluated in
normal acid-base balance andin
NH4C1 acidosis by measuring (a) ammonia
with urine and that addedto
(b) amino acid exchange across the kidney.
acidosis not only urinary ammonia excretion
increased, but also total ammonia production is
0.005) in comparison with controls. By
evaluating the individual role of acid-base parameters,
influencing renal ammonia
production, it was
shown that the degree of acidosis
flow are likely major
the preferential shift of ammonia into
increased and the totalammonia
extracted ratio was higher thanin
inversely correlated with the
of arterial bicarbonate concentration (P<
the same condition, renal glycine and ornithine uptake
the more severe the acidosis, the greater
the renal extraction of these amino acids (P<
0.001). These data indicate thatat
the early stages
of metabolic acidosis,in
production, the mechanisms
the increased glutamine use, whichare
activated and otherammonia
precursors, besides glutamine, are
probably used for
chronic metabolic acidosisinduces an increase in
Portions ofthis paper appeared in anabstract presented
attheVII InternationalCongressofNephrology, Montreal, Canada, 18-23June 1978.
10 August 1981.
ammonia production (1-4) and that this increase
by the kidney (1-6). However, mechanisms
con-trolling renal ammoniagenesis in metabolic acidosis
development of the renal adaptive response
man, urinary ammonia excretion is already
sig-nificantly increased 2 h after the administration of an
load (10-12), but it attains the highest values only
5-6 d after the onset of acidosis (10, 13).
production of ammonia, namely that
plus that added to renal venous blood, was never
man at the initial
stages of metabolic
still uncertain whether theincrease in
urinaryammonia excretion is
merely the consequence
enhanced trapping ofammonia into
urine,or if a
risein renal ammonia
takes place as well.
Moreover, renal utilization ofammonia
of investigations carried out
metabolic acidosis provided
con-flicting results that also dependedon
ar-terial acid-base parameters, which may influenceam-monia excretion in acute
metabolic acidosis have been
the past.An inverse
detected (11, 22,
23); furthermore,an increaseinurine
24). It is poorly understood
abletostimulate renal ammonia
pro-duction. Acidemiaseems to be
the major factor
studies,on the other
both rat renal tissue slices
(26-32)when an acidic
(pH 7.0)is used.
reportedhere were carried out in order to
investigate total renal ammonia
am-monia precursor use in man with normal renal func-tion in an
earlystage of metabolic acidosis. The
was performed by measuringrenal ammonia
individualrole of some factors that may affect ammonia
production,suchasarterialacid-base param-eters, urine pH, and urine
flow,has also been
Patients. 19 hypertensive patients, aged21-60 yr,were
studied. Theirmeanblood pressure ranged from 140 to 160 mmHg.They hadnohistoryorevidence of congestive heart failure, hepaticor pulmonarydiseases,ordiabetes mellitus. Routine laboratory tests, serum and urine electrolyte
con-centration, acid-base measurements, urinalysis, and renal functiontests were normal. Therapidsequenceintravenous
a persistent hyperconcentration of the contrast material. However,nosignificantdifferenceinrenalsize waspresent. All patients were on a standard diet that provided 30-35
kcal and 0.70-0.85 g of protein/kg body weight per d. In
nec-essary for diagnostic purposes to determine plasma renin
activity. Medications werediscontinuedat least 15 dbefore renal vein catheterization. The final diagnosis was benign
essential hypertension. 10 of 19 patients received 159±12
(111-223) mmol/m2 body surface of NH4Cl orally during
the 24hbefore the study. The acid load was given in three fractional doses, the last of whichwasadministered 6 h
be-fore the study. The control and acidotic groupswereevenly
matched for age, sex,bodysurface,and blood pressure.Some
data obtained in 6 of 9 patients presented here as controls were also reported elsewhere (33).
All patients were informed of the nature, purpose, pro-cedure, and possible risks before obtaining their voluntary
Procedure. Patients were studied in the recumbent
po-sition in the postabsorptivestate. A Teflon catheter was
in-serted percutaneously into a peripheral artery to measure
acid-base status and arterial ammonia and AA levels. A
Cobra no. 6 or 7 Scatheter wasthen guided under fluoro-scopic control through a femoral vein to a renal vein. The catheterwaskeptpatentbyintermittentsalineflushes.
Cath-eter position wasascertained visually with image
intensifi-cation before each blood withdrawal.
Anintravenous infusion of Nathiosulphate and Na para-aminohippurate (PAH) wasstartedafter the administration ofapriming dose of PAH (2.5-3 mmol). The infusion was
keptat a constant flowrate (Nathiosulphate 0.8 mmol/min
and PAH0.09 mmol/min)and two or threesequential clear-ance periods of 20 min each were obtained. The correct
position of the catheter in the renal veins was verified by calculating the renalextractionofPAHand oxygen. Oxygen extraction, calculatedas(oxygen.-oxygenv)/oxygen.X 100,
1Abbreviations usedinthis paper: AA,aminoacid;A-V,
arterial-renal venous; GFR,glomerular filtration rate; PAH,
Na para-aminohippurate, RBF, truerenal blood flow.
was used during catheterization as a quick test for checking, with a good approximation, the correct position of the cath-eter. During each clearance period,one setof bloodsamples was obtained simultaneously from a peripheral artery and a renal vein for the measurement of arterial-renal venous
(A-V) differences of ammoniaand AA. Blood sampleswere
taken from renalveinsof bothkidneys. Whensampleswere
undoubtedly obtained from renal veins,PAHextraction and
ammonia and AA A-V differences were not different
be-tweenthe two kidneys.
Bloodwaswithdrawnby heparinized syringes keptin ice.
During the study, urine was collected via acannula under mineral oil and then stored at -25°C in bottles that
Analytical methods. For ammonia determination on
whole blood, samples were deproteinized at +4°C with 0.3 mol/liter Na tungstateand 0.5 mol/liter sulphuric acid
im-mediately after the withdrawal. Theprotein-freesupernate was stored at -25°C and ammonia measured according to Chaney and Marbach (34) within 12h. An eightfold
con-centration of phenol and hypochlorite reagents was used. Thesamemethodwasfollowed for themeasurementof am-monia inurine.
AAwere determined onwhole blood. Proteins were pre-cipitated with cold 0.75 mol/liter perchloric acidwithin60 minfrom the blood withdrawal.Analiquot of supernatewas
neutralized with a buffered solution, stored at -25°C, and used for enzymatic assay of glutamine, glutamate, and
as-partate within 30 h. Another aliquot was stored at -250C and used within 3mofor the determination of 19additional
AA. Specimen storage at -25°C for such a period of time
does not affect AA levels (33, 35). AA analyses were per-formed at least in triplicate by automated ion-exchange
chromatography (Multichrom B AAanalyzer, Beckman
In-struments, Inc.,Fullerton, Calif.). Sodium bufferswereused; the supernatewas neutralized and treated withNasulphite immediately before assay to remove glutathione. This pro-cedure causesa lossof methionine and cysteine. Glutamine was determined enzymatically according to the method re-ported by Lund (36), modified for fluorometer measure-ments (Farrand A 4, Farrand Optical Co., Inc., Valhalla,
N. Y.). Recovery of glutamine, added to whole blood, was
measured for each assay; the recovery ranged from 96.5 to
102%. Glutamate and aspartate were determined according
to Graham and Aprison (37). Aspartate was measured
en-zymatically only in seven controls andin five patients with
NH4Cl-induced acidosis. Becausevaluesobtained enzymat-ically were not different from those provided by ion-ex-change chromatography, only the latterare reportedinthe resultsection. Forenzymatic assays ofglutamine, glutamate and aspartate,samples, blanks, and standards weredone in
triplicate. AA in urine were determined by ion-exchange chromatography on a Multichrom B analyzer after
Glomerular filtration rate (GFR) was measured with the
Na tiosulphate method (38). Renal plasma flow was deter-mined with PAHaccordingto Smith et al. (39).
Blood and urine pH and PaCO2 were estimated at 370C
with PHM 72/BMS 3 apparatus (Radiometer Co., Copen-hagen, Denmark). Blood HCO3 was calculated using the Henderson-Hasselbalch equation. Urine titratable acidity was measured by titration with 0.1 mol/liter NaOH up to
arterial pH. SaO2 was measured with an Hellige oximeter (American Optical Corp., Scientific Instrument Div., Buf-falo,N.Y.). Hematocritwas determined by a microcapillary procedure.
from clearance and extraction of PAH using the Wolf equa-tion(40). True renal arterial bloodflow (RBF) was calculated from renal plasma flow and arterial hematocrit.
Netextraction (+) or net release (-)of individual AA and ammonia by the kidney were calculated by the following formula: M = (Fa X Sa) - Sv
S.),where: M = net uptake or release
(Mmol/min.1.73 m2), Fa = RBF
(ml/min.1.73m2), F,,=urine flow (ml/min- 1.73m2),
Sa= arterial level of metabolite
(,umol/ml),Sv= venouslevel of metabolite
(Mmol/ml),and S,, = urinary level of metab-olite
The total ammoniaproduction was obtained by summing the values for renal venous ammonia release and urinary ammoniaexcretion.
The N balance across the kidney reported here was cal-culated bysubtractingN contributedby individualAA
sig-nificantlyreleasedplusNcontributedby total ammonia pro-duced, from N contributed by individual AA significantly
Patientsandcontrols withNH4Clacidosiswerecompared by analysis of variance by using a completely randomized design. Arandomized block design was applied to the anal-ysisof variance for thepaired data (41). Analysis of simple
regression andcorrelation (41) was used toevaluate the de-pendence of ammonia production and the ammonia-ex-creted/ammonia-produced ratio on NH4CI load, arterial blood acid-base parameters, PNH3inrenalvenousblood and urine, urineflow, and log of urine H+. The same procedure wasfollowed toevaluate the relationships between the fol-lowing variables: renalammoniaproduction andextraction
of glutamine and other AA;ammoniaproduction/glutamine
N extraction ratio and arterial HCO3;exchange of N
con-tributed by glycine plus ornithine and arterial HCO3; and citrulline extraction and arginine output. Analysis of mul-tiple regression (41) was used in order to estimatethe de-pendence ofammoniaproduction (a) and the
(b)on the independent
variables thatshowed a significant simple correlation with (a) and (b). Valuesaregiven as mean±1 SEM.
reports GFR, RBF, blood andurine acid-base
patients under normal
acid-base balance andin
patients with NH4Gl-induced
acidosis. The acid load causeda
of acid administered per squaremeter
urine pH was lowerin
of ammonia excretedwas
dif-ferent from controls.It
follows that totalammonia
the acidotic group
(+63%), and the fraction excreted of totalammonia
directly correlated with the dose of acid load
groups of patients
controls and48±4.5 in
Therewas a direct correlation between renal am-monia
H+(r = 0.807, P
(r= 0.607, P<
flow (r = 0.788, P < 0.001) (Fig. 1). In addition,
inversely correlated with
(r= -0.829, P <
(r=-0.776, P <0.001). However, when
mul-tiple regression analysiswas
carried out witharterial HCO, urine
production correlated significantly
only with arterialHCO
(P <0.01) andurine
ar-terial H+or PaCO2, instead of
production and PNH3 of renal venous blood andurine.
A correlation between the ammonia excretion/am-monia
flow(r = 0.673, P <
0.01) (Fig. 3),the
H+ (r=0.709, P
<0.001) (Fig. 4),and
(r= -0.779; P <
confirmed the dependence of
production ratio upon both urine flow (P
Table II, arterial blood
levels, rates of renal
release, and urinary excretion of 19 free AA
with metabolic acidosis are
were not different in the two
groups, with the
of the arterial histidine level which increased in
normal acid-base balance,
the kidney extracted glutamine, proline, citrulline and
phenylalanine, and released serine, arginine, taurine,
ornithine, tyrosine, threonine,
lysine, histidine, and
glutamate.After 24 h of
different from controls. The only exceptions
glycine and ornithine, which were
by the kidney onlyin
the acidotic group;in
histidine disappeared. Bothin
directly correlated with arginine released
by the kidney (r=
by the kidney
controls and111±5.7 in
well balanced withN
m2in controls and 135±11.6 in
Effects of 24-h Metabolic AcidosisonGFR, RBF,Blood and Urine Acid-baseParameters, UrineFlow,
TotalAmmoniaProduction, and BidirectionalAmmoniaReleaseby the Kidney'
NH! added to
Titratable Urinary therenal, TotalNH: Urinary NH4+ GFR RBF pH. HCO3. UrinepH acidity Urineflow NH4+ veins production TotalNH+production
inl! '''mi/mm. mmol/ peq/min m /min/ MnO/ jmol/ ;MO,/
min- 1.73 m' liter 1.73m2 1.73m' min- min- min-1.73m2 1.73m' 1.73m2 1.73m'
Controls(9) 145±6.1 1098±79.0 7.41±0.010 22.6±0.69 5.58±0.134 15.6±2.75 1.83±0.227 22.5±1.99 18.2±1.45 40.6±2.34 0.55±0.030 24-hNH4CI
acidosis(10) 139±7.3 956±69.8 7.33±0.010" 15.1±0.98" 4.74±0.058" 37.8±1.50" 3.09±0.492t 51.5±6.05" 14.6±1.33 66.1±5.84§ 0.76±0.027"
Abbreviationsusedinthis table:GFR,glomerular filtrationrate; RBF, truerenal blood flow.
* Values are given as mean±SEM.
Significanceofdifferencefrom thecorresponding valueincontrols:
supplied by glutamineextractionwasnot
changed despite theincrease in
ex-traction ratio doubled andwas
with the log
of arterial HCO- (r=
contributed by glycine and ornithine took
Finally, the exchange ofN
by glycine and
presented here demonstrate thatin
already known (10-12),
y=26.399 + 11.080 x (r=0.788; P< 0.001)
0 o 0
051.5 2.5 3.5 4.5 5.5
ml / min .1.73 m2
FIGURE1 Relationships between total renalammonia productionand urineflowin 9patients
withnormal acid-base balance (solid circles) andin10patientswith 24-hNH4Clacidosis (open
RE 40 2
-< 20 0
y=117.845 -3.418 x (r- -0.829; p< 0.001)
10 13 16 19 22 25 28
ARTERIAL HC(I CONCENTRATION
FIGURE 2 Relationships between total renalammonia productionand arterial HCO3
concen-tration in9 patients with normal acid-base balance (solid circles) and in 10patients with
24-h NH4Clacidosis (open circles).
y=0490 +0.069 x (r-0.673; P'0.01)
0.5 1.5 2.5 3.5 4.5
ml /min 1.73 m2
FIGURE 3 Relationships between the urinary ammonia excretion/total ammonia production
ratio andurineflowin9patientswith normal acid-basebalance(solid circles)and in 10patients with24-h NH4C1 acidosis (open circles).
U 0 0 a:
y=O.O50 +o.189 x (r=0.709; p<O.oo1)
-0.2 0 0.2 0.4 0.6
LOG URINARY H* C iJmol / liter
1.2 1.4 1.6
FIGURE4 Relationships between the urinary ammonia excretion/total ammonia production
ratioandthe logofurinaryH+concentration in9patients with normal acid-base balance(solid circles)and in 10 patients with 24-h NH4Cl acidosis (open circles).
but also total renalammonia
Thus,metabolic acidosis induces a precocious
of buffer becomeavailableto meetthe
needto excrete fixed
Recent studies in the isolated
have indicated thatthe fall of urine pH, rather than the decrease of HCO- in the
perfusion fluid,is the critical
stimulusfor increased ammonia
theonset of metabolic
flow,rather than the fall ofurine pH,arethe major factors
increase in ammonia
production.The importance of
acidemia hasalso been outlinedin the intact ratwith
meaningless (17).Moreover, nocorrelation was
productionand urine pH
both in dog and in man under normal acid-base bal-ance and in chronic
(1,42,43). It has been shown that
linearlycorrelated with extracellular
pH (44). Thus, acidemia may induce a fall of pH
within tubular cells and consequently, promote an
A quite novel
findingis the apparent role of urine
flow as a stimulus for ammonia
hypothesizethatinthis condition the
increased urine flow and the consequent
washout ofurinary ammonia are more
the fall in urine pH in
am-monia from cells into tubular
lumen, hence,in low-ering ammonia concentration in tubular
production,on the other
hand,seems to be
independentofPNH3in renal venous
The simultaneous measurement of ammonia
ex-creted withurineandthat addedtorenalvenous
carriedout in this research allows oneto estimate the actual role ofurine pHand urine flow in
acidosis,the increase in both urine
and urine flowis
shift-ing of ammonia into urine. In
fact, the fraction
ex-creted of total ammonia
propor-tionallyto urine flow and to the
findingin the present
man, at the onset of metabolic
gluta-mine extraction is not different from that measured
in normal conditions in spiteofa 63% increase in
production.In addition the ammonia
produc-tion/glutamineN extraction ratio rises
proportionallyto the fall of arterial HCO-; these data indicate that the
discrepancybetween ammonia production and z
o o - 8 t- 8 o o
+~~-Hio o o o o o 0o
o cot t0>NO 04ut104cqtsc c qe
O VCOt O DO iOO_OcO O _U4cf
-o e_o o o
0 -4000 000 0
-. . . .
0 0 14I_ tC _ CD CD
I4 I6 ++4
+1 +1 +1
. .i.t. .t.
CO - 1' k6 CO5 - -
-- -4 -+l +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1
00 co -_ 00t-r a C N c tCo V 0
4 oi 0r- 0--- - cO cO cD COD
+ + + + +
o0-o > o cqUG0 _ _ o
0-C4- 00-0 t- t- kf
+l +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1
0 0 0o 0 0 -4 CD -.4 0) t -4
-o~0~-e~I~cci O 6 C4 t6 C4 O its - e' O
C _ _-1 + + +
+1+1 +1 +1 +1+1+1 +1 -H +1 +1+1
+1~+1+1+1+~~ 1+1+ +1 + +1
I ++ + I+ I+
.40.0 **Oi g
CD 00 00a0
+1 +1 +1 +1 +1 +1 +1
_; C6 i
IVr to t- T5 co 14 0 9 t--'-bu:0
t-o6 -4 is. _ C6 F . U:1f~CDoa
_ b _ _ CUC_ U 0 0CO1tU
+ +1 +1 +1 +1 +1 +1+1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 uz ut Cb N cq >_ CO 0cn a _ o e o q oDa 0
0j04 1- kf6 -4-r kf6 c
CS0> t O Ci Ut _ e _ _ U b CD r o
00 cq 0 t
_- _V _ o oo 0 gC!F za tc ebt
-4 4 t o - 4-4
01->~~~~~~~~_itc0N O6 g tzt- ei ci
0 O c o-gNt CO 0 kC D
_u"~t o>ac~ 0 _tc t0.~O tr_o
Ck00Co____u:_>cQCC D C 0) t 0 .e CO
i 0VV V V0V
I o _*
Renal Production ofAmmoniaand Utilization of Glutamine, Glycine,and OrnithineinControls and in Patients with 24-h MetabolicAcidosis*
TotalNH! NH:production Gly N + Om N
production GlnN extraction GlnNextraction GinNreabsorbed exchange gsmoJ/min 1.73m2 ismol/min-1.73mO pmd/min * 1.73 m' smnd/min-1.73 n'
Controls(9) 40.6±2.34 67.0±9.16 0.68±0.083 150.2±10.73 -6.6±4.03 24-h
Abbreviationsused in this table: Gln,glutamine; Gly,glycine;Orn, ornithine. Values aregiven as mean±SEM.
I Determined onlyin sevensubjects.
§Significantly different fromthecorrespondingvalueincontrols,P<0.005.
along with the degree
of acidosis. Conversely,in
NH4Cl-in-duced chronic acidosis, theammonia
extractionratio is not
different from that
subjects under normal acid-base
calculate from data reported
Furthermore, assuming that
alanine and glutamatein
by renal glutamine extractionis
formation. Thusin acute
acid-basecon-ditions and to chronic
produced by the kidneycan
glutamine extracted alone,even
complete utilization ofN
in man,in an
early stage of metabolicacidosis,
maintain its role as the nearly
The reason why renal glutamine extraction does not
of metabolic acidosis is so far
y-499 -3.176 X (r -0.750;P< 0.001)
1.0 1.1 1.2 1.3 1A
LOG ARTERIAL HCX) CONCENTRATION mmol / liter
FIGURE 5 Relationships between the renalammoniaproduction/glutamineNextraction ratio and thelog of arterial
HCOiconcentration in 9patients with normal acid-base balance (solid circles) and in 10 patients with 24-h NH4Cl acidosis (open circles).
+20 0 z
y-67659 - 3 272 X
mm FIGURE 6 Relationships betwe contributed byglycineandornit centration in nine patients wit
(solid circles) andin sevenpatie
obscure. Glutamine supply
changed, because theamoun
by farin exce
patients.Conversely, acute acidification modifies the
exchange of some AA across the kidney, that is, a
sig-nificant glycine and ornithine extraction takes place.
Particularly noteworthy is the effect of acidosis in
in-fluencing renal glycine and ornithine metabolism. The
the acidosis, the greater is the extraction
contributed by glycine and ornithine. It is
contributed by glycine and
nithine extracted may cover the amount of ammonia
not accounted for by the glutamine extracted. The data
presented here cannot provide direct evidence that
glycine and ornithine extracted are used for ammonia
acidosis. However, glycine can be
used for ammoniagenesis in the intact dog (51, 52),
produce ammonia by rat renal
tical slices (53) through conversion to glutamate. Two
other AA, namely proline and citrulline, are extracted
by the kidneyin
acidosis at the same ratesas in
citrulline cannot be considered an
precursor inasmuch as thisAA is
kidney for arginine production in normal acid-base
54) and probablyin
patients with 1-d
acidosis studied here;in
ob-servations). Proline may bean
through conversion to glutamate; however, because
1821 24 27 proline extraction does not increase in acute acidosis,
conclusion, studies reported here demonstrate
en the renal exchange of N that in man the
HCO-con- to mabolic
thnormal acid-base balance to
metabolic acidosis is a precocious event. At an early
stage of acidosis, acidemia and increasedurine
major role in stimulating ammonia
pro-duction. In this stage, the adaptive mechanisms
tubular cellsis not
sponsiblefor the increased renal
glutamine filtered andare
of glutamine extracted
tivated; other substrates, besides glutamine extracted
which the effect of pHon
from the arterial blood,are
probably utilized for
agree that bothmonia
production from glutamine and
doesnot stimulate rat renal
188.8.131.52.) (46-49) and
dehydrogenase (E.C. 184.108.40.206.) (50)ac-tivities.
All the data obtainedinvitro
failure of renal
in-crease in man in
metabolic acidosis.Because in
condition, despitethe increased ammonia
pro-duction, glutamineextraction is
glutamate probablycannot be taken into account be-cause
the release of thisAA is
The authors wish to thank Mr. Mario Bruzzone for his
ex-cellent technical assistanceand Mrs. FrancescaTincani for
preparation ofthe manuscript and the figures. Some of the resultsreportedinthis paper werediscussedwith Dr. Robert G. Narins(Department of Medicine,SectionofNephrology, TempleUniversity, Philadelphia, Pa.) towhomthe authors are grateful foradvice and criticism.
Thisstudy wassupported by grants39/74.00860.65, 42/
77.01578.65, and 95/78.02759.65 from the Consiglio
Na-zionale delle Ricerche under the cooperative agreement
be-tween Italy and the United States of America.
kid-neyduringthe prolongedadministration of ammonium
chloride. J. Clin. Invest. 42: 263-276.
2. Tizianello, A.,G. Deferrari, G. Garibotto, and G.
Gur-reri. 1978. Effects of chronic renal insufficiency and
metabolic acidosis on glutamine metabolism in man.
Clin. Sci. Mol. Med. 55: 391-397.
3. Pitts, R. F., J. DeHaas, and J. Klein. 1963. Relation of
renalamino and amide nitrogenextraction to ammonia
production.Am. J. Physiol. 204: 187-191.
4. Relman, A. S.,and S. Yablon. 1978. Theregulation of ammonia production in the rat. In Biochemical
Ne-phrology, W. G. Guder and U. Schmidt, editors. Hans
Huber Publ., Bern, 8: 198.
5. Van Slyke, D. D., R. A. Phillips, P. B. Hamilton,R. M.
Archibald,P.H.Futcher, andA.Hiller.1943.Glutamine as source material of urinary ammonia. J. Biol. Chem.
6. Welbourne,T.C. 1973. Lerole dureindans laregulation de la glutaminemie. L'Union Medical du Canada. 102: 1451-1457.
7. Pitts, R.F. 1973. Production andexcretionofammonia
in relation toacid-baseregulation. InRenalPhysiology. J. Orloff and R. W. Berliner, editors. American
Phys-iological Society, Washington. 15: 455-496.
8. Stoff, J. S., F. H. Epstein, R. Narins, andA. S. Relman. 1976. Recent advances in renal tubular biochemistry.
Annu. Rev. Physiol. 38: 46-68.
9. Tannen,R. L.1978.Ammoniametabolism.Am.J. Phys-iol.235: F265-F277.
10. Sartorius,0. W., J. C.Roemmelt,and R. F. Pitts. 1949.
Therenal regulation of acid-base balance in man. IV.
The nature of the renal compensations in ammonium
chloride acidosis. J. Clin. Invest. 28: 423-439.
11. Wrong, O., and H. E. F. Davies. 1959. The excretion
of acid in renal disease. Q. J. Med. 28: 259-313. 12. Tannen,R. L. 1969. TherelationshipbetweenurinepH
and acid excretion-the influence of urine flowrate. J. Lab. Clin. Med. 74: 757-769.
13. Simpson, D. P. 1971. Control of hydrogen ion
homeo-stasisand renal acidosis.Medicine. 50: 503-541. 14. Denis, G., H. Preuss, and R. F. Pitts. 1964. The pNH3
of renal tubular cells. J. Clin. Invest. 43: 571-582. 15. Addae, S. K., and W. D. Lotspeich. 1968. Relation
be-tweenglutamine utilization andproductioninmetabolic acidosis. Am.J. Physiol. 215: 269-277.
16. Weiss,F. R., andH.G. Preuss. 1971.Glutamate metab-olism andammoniaproductionindogkidneys.Nephron.
17. Emmett, M., J.Rascoff,A. S. Relman,andR. G. Narins. 1976. Effects of acuteacid-base changeson in vivo rat
renal NH3 synthesis. Proc. Annu. Meet. Am. Soc.
Ne-phrol. 9: 96.
18. Fine, A.,F. I. Bennet, and G. A. 0.Alleyne. 1978. Ef-fectsofacuteacid-base alterationsonglutamine metab-olism and renal ammoniagenesis in the dog. Clin. Sci.
Mol. Med. 54: 503-508.
19. Goldstein, L., and J.M. Boylan. 1978. Renal mitochon-drialglutamine transport and metabolism: studies with
arapid-mixing,rapid-filtrationtechnique.Am.J. Phys-iol. 234:F514-F521.
20. Hughey, R.P.,B. B. Rankin, and N. P. Curthoys. 1980. Acute acidosis and renal arteriovenous differences of glutamineinnormal and adrenalectomized rats. Am.J. Physiol. 238: F199-F204.
21. Vinay, P.,E.Allignet,C. Pichette,M.Watford,G.
Lem-ieux, andA.Gougoux. 1980.Changesinrenalmetabolic
profile and ammoniagenesis during acute and chronic
metabolic acidosis indogand rat.Kidney Int. 17: 312-325.
22. Stanbury, S. W., and A. E. Thompson. 1951. Diurnal variations in electrolyte excretion. Clin. Sci. 10: 267-293.
23. Clarke,E., B. M. Evans, I. Macintyre,andM. D. Milne.
1955. Acidosis in experimental electrolyte depletion. Clin. Sci. 14: 421-440.
24. Woeber, K. A., E. L. Reid, I. Kiem, and A. G. Hills. 1963. Diffusion of gases out of the distal nephron seg-ment in man. I. NH3. J. Clin. Invest. 42: 1689-1704. 25. Tannen, R. L., and B. D. Ross. 1979. Ammoniagenesis
by the isolatedperfused rat kidney: the critical role of urinary acidification. Clin. Sci. 56: 353-364.
26. Pagliara,A. S., and A. D. Goodman. 1970. Relation of renal cortical gluconeogenesis, glutamate content, and production ofammonia. J.Clin. Invest. 49: 1967-1974.
27. Goodman, A. D. 1973. Effectof acid-basechanges and dehydrationonrenalmedullary production ofammonia. J. Lab. Clin. Med. 81: 905-918.
28. Alleyne, G. A. O., and A. Roobol. 1974. Regulation of
renal cortex ammoniagenesisI.Stimulationof renal
cor-tex ammoniagenesis in vitro by plasma isolated from acutely acidotic rats. J. Clin. Invest. 53: 117-121.
29. Preuss, H. G., 0. Vavatsi-Manos, L. Vertuno, and K. Baird. 1974. The effects of pH changes on renal
am-moniagenesis in vitro. Proc. Soc. Exp. Biol. Med. 146:
30. Cartier, P., P. Belanger, and G. Lemieux. 1975. Char-acteristics of in vitro ammonia and glucose production by dog kidney cortex. Am. J. Physiol. 228: 934-943. 31. Relman, A. S., and R. G. Narins. 1975. The control of
ammonia production in the rat. Med. Clin. N. Am. 59: 583-593.
32. Tannen, R. L., andA. S. Kunin. 1976. Effect ofpH on ammonia production by renal mitochondria. Am. J.
Physiol. 231: 1631-1637.
33. Tizianello, A., G. Deferrari, G. Garibotto, G. Gurreri,
andC. Robaudo. 1980. Renalmetabolismofamino acids
andammonia insubjects withnorma; renal function and
in patientswith chronic renalinsufficiency. J. Clin.
In-vest. 65: 1162-1173.
34. Chaney, A. L., and P. E. Marbach. 1962. Modified
re-agents for determination of urea and ammonia. Clin.
Chem. 8: 130-132.
35. Perry, T. L., and S. Hansen. 1969. Technical pitfalls leading to errors in the quantitation of plasma amino acids. Clin. Chim. Acta. 25: 53-58.
36. Lund, P. 1974. L-glutamine: determination with
glu-taminaseand glutamate dehydrogenase. In Methods of enzymaticanalysis. H. U.Bergmeyer, editor. Academic
Press., Inc., New York. 2ndedition. IV: 1719-1722.
37. Graham, L. T., Jr., and M. H. Aprison. 1966. Fluoro-metricdeterminationof aspartate, glutamate and gamma-amino-butirate in nerve tissue using enzymic method. Anal. Biochem. 15: 487-497.
38. Brun, C. 1950. Thiosulphate determination in kidney
function test. J. Lab. Clin. Med. 35: 152-154.
39. Smith, H. W., W. Goldring, and H. Chasis. 1938. The measurement of tubularexcretorymass,effectiveblood
flow and filtration rate in the normalhuman kidney. J. Clin. Invest. 17:263-266.
40. Wolf,A. W. 1941.Total renal bloodflowat any urinary flow or extraction fraction. Am. J. Physiol. 133:
methods. Iowa State Univ. Press, Ames, Iowa, 6th
42. Hills, A. G., and E. L. Reid. 1966. Renal ammonia
bal-ance. A kinetic treatment. Nephron. 3: 221-256. 43. Hills,A. G., andE. L. Reid. 1970. PCO2 and PNH3 in
mammalian kidney and urinary tract related to urine pHandflow. Am.J. Physiol. 219: 423-434.
44. Struyvenberg, A., R. B. Morrison, and A. S. Relman. 1968. Acid-base behavior of separated canine renal
tu-bule cells. Am. J. Physiol. 214: 1155-1162.
45. Stone,W.J., andR. F.Pitts. 1967.Pathways ofammonia
metabolism in the intactfunctioning kidney of the dog. J. Clin. Invest.46: 1141-1150.
46. Leonard, E., andJ.Orloff. 1955.Regulation ofammonia
excretion in therat. Am.J. Physiol. 182: 131-138.
47. Rector, F. C., Jr., D. W.Seldin, and J. H. Copenhaver. 1955. Themechanism of ammonia excretion during
am-moniumchloride acidosis. J. Clin. Invest. 34: 20-26. 48. Alleyne, G. A.O., and G.H.Scullard. 1969. Renal
met-abolic response to acid-base changes. I. Enzymatic
con-trol of ammoniagenesis in the rat. J. Clin. Invest. 48: 364-370.
49. Adam, W., and D. P. Simpson. 1974. Glutamine trans-port in rat kidney mitochondria in metabolic acidosis.
J. Clin.Invest. 54: 165-174.
50. Kunin, A. S., and R. L. Tannen. 1979. Regulation of glutamate metabolism by renal cortical mitochondria.
Am.J. Physiol. 237: F55-F62.
51. Pitts, R. F., L. A. Pilkington, andJ. DeHaas. 1965. N'5
tracerstudies onthe origin ofurinary ammonia in the acidotic dog, with notes on the enzymatic synthesis of labeled glutamic acid and glutamine.J.Clin.Invest. 44: 731-745.
52. Pitts, R. F., and L. A. Pilkington. 1966. The relation between plasmaconcentration ofglutamineandglycine and utilization of their nitrogens as sources or urinary
ammonia. J. Clin.Invest. 45: 86-93.
53. Krebs, H. A. 1933. Untersuchungen uber den Stoff-wechsel der Aminosauren im Tierkorper. Z. Physiol. Chem.217: 191-227.
54. Rogers, Q. R., R. A. Freedland, and R. A. Symmons. 1972. In vivosynthesis and utilization of arginine in the