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Yale UniversityEliScholar – A Digital Platform for Scholarly Publishing at Yale
Yale Medicine Thesis Digital Library School of Medicine
1980
Effect of ketone bodies on the renal excretion ofammoniaGary DesirYale University
Follow this and additional works at: http://elischolar.library.yale.edu/ymtdl
This Open Access Thesis is brought to you for free and open access by the School of Medicine at EliScholar – A Digital Platform for ScholarlyPublishing at Yale. It has been accepted for inclusion in Yale Medicine Thesis Digital Library by an authorized administrator of EliScholar – A DigitalPlatform for Scholarly Publishing at Yale. For more information, please contact [email protected].
Recommended CitationDesir, Gary, "Effect of ketone bodies on the renal excretion of ammonia" (1980). Yale Medicine Thesis Digital Library. 2519.http://elischolar.library.yale.edu/ymtdl/2519
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https://archive.org/details/effectofketoneboOOdesi
Effect of Ketone Bodies on the Renal
Excretion of Ammonia
by
Gary Desir
BA New York University 1976
A thesis submitted to the faculty of the Yale
School of Medicine in partial fulfillment of
the requirements for the degree of doctor of
medicine 1980
To my parents
Gerard and Claude Desir
Acknowledgements
I wish to thank the following persons who have helped me
complete this work;
Dr Ralph DeFronzo,my supervisor, whose criticism was most
stimulating and whose advice was invaluable; It was a
pleasure working with him;
Dr Fred Wright who carefully reviewed the manuscript;
Lois Mishiwiec who was most helpful in taking care of the
patients;
Nadia Myketey,supervisor of the renal laboratory,whose
careful guidance made working in the laboratory a pleasure
Yihfen Wu for expert technical assistance;
Deborah Dyett,my wife,for constant,much needed support.
TABLE OF CONTENTS
Page
Introduction 1
Literature Review 2
Renal Ammoniagenesis 2
Biochemistry of renal ammoniagenesis 3
Sites of ammonia production and excretion 6
Mechanism of ammonia secretion 7
Source of renal ammonia 8
Regulation of ammonia production and
secretion 9
Ketone bodies metabolism 19
Regulation of ketone uptake 19
Effects of ketones on glucose metabolism 20
Effects of ketones on amino acids
release 21
Methods 22
Results 28
Discussion 34
Summary 45
Appendix 46
References 76
1
I INTRODUCTION.
The urinary excretion of ammonium increases markedly
during both diabetic (1,2) and starvation (3) ketoacidosis.
The rise in renal tubular hydrogen ion concentration that
results from the increased production of ketoacids is be¬
lieved to stimulate ammoniagenesis (4). Surprisingly how¬
ever, a significant decrease in urinary ammonium excretion
has been reported during acute ketone infusion in chronic¬
ally acidemic dogs (5). The fall in ammonium excretion was
not related to changes in urinary pH or in systemic acid
base status. It was, however, accompanied by a striking
drop in glutamine extraction by the kidney. Whether hyper-
ketonemia also inhibits ammonium excretion in man is not
known. Consequently, the present study was carried out to
determine the effects of sodium 3-hydroxybutyrate on the
urinary excretion of ammonium in humans made chronically
acidemic by ammonium chloride loading.
2
II. LITERATURE REVIEW.
A. Renal ammoniagenesis.
In man ingesting a normal protein intake, 1-1.5 meq/kg
of strong acids are produced daily. This results primarily
from the generation of phosphoric and sulfuric acid during
the metabolism of phospholipids and sulfur containing amino
acids. The hydrogen ion is initially buffered by extra¬
cellular bicarbonate and the accompanying anion is transport
ed to the kidney and is filtered. Those filtered anions
can then serve as proton acceptors for distally secreted
hydrogen ions. These titratable acids (TA) account for about
half the amount of hydrogen ion which is excreted daily. The
remaining half of daily hydrogen ion production is excreted
with ammonia which because of its high pKa is an excellent
urinary buffer. The excretion of titratable acids and
ammonium permits the reabsorption of fixed cations and the
restoration of the body's buffer stores.
During metabolic acidemia the major increase in hydro¬
gen ion excretion is accounted for by an increase in ammonia
production by the kidney. As a buffer, ammonia is unique
for several reasons (6): 1) Unlike phosphate which is drawn
from cells and bones only at the expense of interference
with function and structure, ammonia can be supplied from
waste nitrogen and its production and release are metabolic-
ally inexpensive. In experimental acidemia, ammonia pro-
3
duction increases markedly and the ratio of titratable acids
to ammonium ion falls from 1:2.5 to 1:5 - 1:10. The ability
of titratable acid excretion to increase during acidemia is
limited by the glomerular filtration rate (GFR) and the de¬
pletion of total body phosphate stores. 2) In solution,
an equilibrium exists between ammonia (NH^) which is read¬
ily diffusable and ammonium ion (NH^t)which is poorly diffus
able. The ratio of NH^/NH^+ is determined by the pH of the
solution. Acidification increases and alkalinization de¬
creases the ratio. It follows that an acid urine acts as
a sink for trapping NH^ in its cationic form thereby pre¬
venting its back diffusion into the renal tubular cell and
thereby enhancing its secretion into the urine. 3) NH^
production is capable of increasing 10-20 fold in chronic
metabolic acidemia unlike titratable acids (TA) excretion
which is limited by GFR.
Biochemistry of renal ammoniaqenesis.
Approximately 90% of ammonia produced in the kidney
is derived from glutamine. Other amino acids such as alanine
glycine and histidine account for 10% of renal ammoniagene-
sis (7). The metabolism of glutamine has been extensively
studied. In the rat, the pathway of glutamine metabolism
has two parts: The first consists of the reactions leading
to the formation of ammonia and the second is concerned
with the disposal of a-ketoglutarate, the carbon skeleton
4
of glutamine, (figure 1). a-ketoglutarate enters the tri¬
carboxylic acid (TCA) cycle where it is converted to carbon
dioxide and water. Alternatively it can be transported to
the cytosol where it is converted to phosphoenolpyruvate
by phosphoenolpyruvate carboxykinase (PEPCK) and then
toglucose via several reactions. As will be discussed
later, gluconeogenesis may play an important role in the
control of ammonia production.
Ammonia can be produced from glutamine through sever¬
al reactions (Figure 1):
a) Deamidation of glutamine:
, , . glutaminase I . , . , glutamine __glutamate + NH^
b) Deamination of glutamate:
! , glutamic dehydrogenase . , . , . , *,TT glutamate __d J^ a-ketoglutarate +NH^
c) Transamination followed by hydrolysis:
. , , . . _ glutamine keto acid , glutamine + pyruvic acid __^ alanine +
transaminase aTketogl.
a-ketoglutaramate ^ amidase a_]<etoglutarate + NH^
The deamidation of glutamine is catalyzed by glutaminase I.
In 1935 Krebs (8) described enzymes in liver and kidney that
hydrolized glutamine to ammonium and glutamate. Since the
pH optimum of the liver enzyme (8.8) differed from that of
the kidney, he inferred that they were different enzymes
and anticipated the discovery of glutaminase I (kidney) and
glutaminase II (liver). Glutaminase I exists as two iso¬
enzymes; one requires phosphate for activity and is termed
phsophate dependent glutaminase (PDG); the other does not
and is called phosphate independent glutaminase (PIG) (9).
For the most part deamidation is catalized by (PDG). This
isoenzyme has been well characterized: it is located in the
inner mitochondrial membrane (10), has a pH optimum between
7.8 and 8.5, is heat labile, is inhibited by ammonia and
glutamate and is activated by phosphate, sulfate and several
TCA cycle intermediates (11). PIG also catalizes the deamida¬
tion of glutamine. Its pH optimum is between 7.4 and 7.6;
it is heat labile and is inhibited by its end products (12,
13) .
Unlike deamidation, much less is known about the de¬
amination reaction which is believed not to play a major role
in renal ammoniagenesis (10). This reaction involves an
oxidative process and is catalized by glutamate dehydrogenase
(GD) a mitochondrial enzyme. GD is inhibited by a-ketogluta-
rate (14,15) and is activated by an elevated NAD+/NADH ratio
(16) .
Significant species difference are known to exist
among the reactions. Although both deamidation and deamina¬
tion are known to occur in both rats and men, transamination
takes place only in rats. Furthermore, transamination has
been shown to be important in renal ammoniagenesis in the
rat. Welbourne has demonstrated that in the normal rat kid¬
ney this reaction contributes 70% of the total ammonia pro¬
duction (17). Glutamine glutamyl transferase (GT) catalyzes
6
the transamination reaction. This enzyme complex is found
in the cytosol and is bound to the endoplasmic reticulum
(18) is non specific, is competitively inhibited by aceta-
zolamide, exhibits both hydrolytic and transferring activit¬
ies and catalyzes the following reactions:
glutamine glutamyl trans. 2 glutamine ^ 2 NH + y-glutamyl
glutamine + hydroxylamine GT
glutam.
NH^ + glutamyl hydroxamate
As noted above, in the normal rat kidney, those reactions
account for most of the ammonia produced. However, in the
acidotic rat, the deamination of glutamate and the deamida¬
tion of glutamine are responsible for 80% of total ammonia
production (17).
Sites of ammonia, production and excretion.
Since the glutaminase enzymes are of major impor¬
tance in ammonia production, their localization in the
nephron has been used to identify the sites of ammoniagenesis.
Goldstein et al (19,20) showed that although glutaminase I
activity was highest in the renal cortex and inner medulla
in rats, enzyme activity in the collecting duct was more
than sufficient to account for all the ammonia excreted in
the urine. They concluded that ammoniagenesis was primarily
a function of the collecting duct. However, several ex¬
periments suggest that the proximal and perhaps the distal
tubule as well, contribute significantly to ammoniagenesis
7
in the acidemic rat. The major increase in glutaminase I
activity following metabolic acidemia occurs in the renal
cortex, not in the renal medulla (21). Furthermore, Seyama
(22) demonstrated that the increase in glutamic dehydro¬
genase (GD) activity in acidosis is largely associated
with epithelial cells of the proximal convoluted tubule.
Micropuncture studies done by Glabman, Klose and Giebisch
(23) conclusively demonstrated that ammonia secretion
actually occurs throughout the entire nephron. Hayes (24)
confirmed those findings and documented that 70% of urinary
ammonia was secreted by the proximal tubule and that most
of the remaining 30% was added by the distal tubule. It
should be noted, however, that the sites where ammonia is
secreted need not be identical to those at which it gains
final access to the urine. Indeed, Pitts (25) has provided
evidence that considerable recycling of NH^ occurs in the
medullary interstitium. Ammonia produced and secreted in
the proximal tubule diffuses out of the loop of Henle into
the papillary interstitium and eventually gains entry into
the collecting duct cell from where it can diffuse into the
tubular lumen. In summary, the available evidence suggests
that in acidosis most of the production and secretion of
ammonia is carried out by the proximal tubule.
Mechanism of ammonia secretion.
It is now well established that ammonia secretion is
8
passive and effected by a mechanism of non ionic diffusion
(26, figure 2). The renal tubular membrane is fully per¬
meable to ammonia and this enables the free base to diffuse
down very small concentration gradients into acid urine where
it is trapped as ammonium ion. Since diffusion occurs over
small distances, it is never a limiting factor in ammonia
secretion.
Source of renal ammonia.
The various sources of renal ammonia are shown in
figure 3. Van Slyke et al (28) were the first to show that
in acidemic dogs glutamine is extracted by the kidney and
that its amide nitrogen alone could account for approxi¬
mately 2/3 of the total amount of ammonium excreted. During
metabolic acidemia in men (29) and in dogs (30), glutamine
extraction by the kidney increases and greatly exceeds that
of any amino acids. Using labeled glutamine, several groups
(7,31-35) have demonstrated that the amide nitrogen of
glutamine accounts for approximately 43% and the amino nitro¬
gen for another 18% of the excreted ammonium. About 35% of
the ammonium excreted comes from arterial blood filtered by
the glomeruli. Only a small percentage, approximately 4%
of urinary ammonium is derived from the amino nitrogen of
alanine and glycine. Thus about 90% of renal ammonia pro¬
duction is derived from the metabolism of glutamine.
9
Regulation of ammonia production and secretion.
During chronic metabolic acidemia, ammoniagenesis in¬
creases by 10-15 fold and large amounts of ammonium are ex¬
creted in the urine. The rate at which ammonium is excreted
is determined by three factors: 1) the acidity of the urine
2) the rate of urine flow and 3) the amount of ammonia pro¬
duced by the renal tubular cell. As discussed previously,
the permeability of cellular membranes to ammonia is so
great that diffusion is never a limiting factor in ammonium
excretion. Pitts (36) showed that in normal dogs and in
dogs made acutely acidemic, ammonium excretion increased
significantly although ammonia production did not change.
The increase in excretion was found to be negatively
correlated with urine pH. Since hydrogen excretion in¬
creases markedly during metabolic acidemia, in the tubular
fluid ammonia is immediately converted to ammonium ion.
A favorable gradient for anmonia diffusion is thus maintained.
The relationship between urine flow rate and ammonium ex¬
cretion is more complex. If the urine is strongly acidic,
ammonia secretion is independent of urine flow (37). How¬
ever, if the urine is weakly acidic or alkaline, it be¬
comes directly proportional to urine flow. Although it is
clear that urine pH and flow rate are important variables,
during chronic metabolic acidemia the rate of ammonia pro¬
duction is the primary determinant of ammonium excretion
(38) .
10
Several factors appear to affect renal ammoniagenesis:
a) plasma concentration of glutamine, b) renal blood flow,
c) transport of glutamine, d) glutaminase activity, e) cellu¬
lar concentration of hydrogen ion, f) intracellular concen¬
tration of potassium, g) intracellular concentration of
glutamate and NAD+ NADH ratio, h) concentration of ammonia,
i) concentration of a-retoglutarate, j) renal gluconeogene-
sis, k) availability of oxidizable substrates.
A) Effect of plasma concentration of glutamine on NH^
production
During chronic acidemia the extraction of glutamine,
the major precursor of ammonia, increases significantly.
However, the plasma concentration of glutamine remains
constant since the liver,which is the major source of ex-
trarenal glutamine,increases its production (39,40). In
the normal dog, plasma glutamine is increased and ammonia-
genesis is stimulated by intravenous infusions of glutamine
(41). This suggests that the amount of precursor delivered
to the kidney determines, how much ammonia is producdd.
B) Effect of renal blood on NH^ production
Olert and Nagel (42) have postulated that although
renal glutamine delivery varied with GFR, substrate supply
was independent of renal blood flow since the amount of
glutamine filtered and reabsorbed in the normal dog was
11
sufficient to account fully for the amount of ammonia
produced. In metabolic acidemia, however, glutamine uptake
increases markedly and filtered glutamine is not sufficient
to meet the demand for ammonia producing substrate. Con¬
sequently, in metabolic acidemia large amounts of gluta¬
mine are actively taken up at the peritubular surface of
the renal tubular cell (43,30). Lemieux et al (44) docu¬
mented that in the acidemic dog, 60% of ammonia produced is
derived from glutamine extracted at the antiluminal surface
Thus, under conditions of metabolic acidemia, renal ammonia
production becomes very much dependent upon renal blood
flow.
C) Effect of glutamine transport on NH^ production
Although the delivery of glutamine to the kidney and
its transport into the peritubular cell cytoplasm are
essential for ammoniagenesis, other factors are also
important. The availability of glutaminase I plays a
primary role in the regulation of ammonia production.
Since this enzyme is located within the mitochondrion,
(45) glutamine must reach the mitochondrial inner membrane
and changes in membrane permeability can affect ammonia pro
duction. The presence of a variety of transport systems
for ions, substrates and products across the inner mito¬
chondrial membrane has been demonstrated. There are group
specific transport systems for-dicarboxylic acids (malate.
12
malonate, succinate), tricarboxylic acids (citrate, iso¬
citrate), a-ketoglutarate and for glutamate (46). Simpson
demonstrated an increase in glutamate formation and ammonia
production in mitochondrial and submitochondrial preparations
from acidemic dogs in the absence of changes in glutaminase
I activity (47). He has also provided evidence suggesting
that passive movement of glutamine across the inner mito¬
chondrial membrane did not occur and has postulated that a
carrier for glutamine exists in the inner membrane (48).
Crompton et al have shown that glutamine transport is rate
limiting in the deamidation reaction by intact mitochondria
from hog kidney (49). It has/thusf been suggested that the
rise in ammoniagenesis observed in metabolic acidemia was
due to an increase in glutamine transport by the carrier
transport located within the inner mitochondrial membrane
(48) .
Glucocorticoids may modulate the activity of the carrier
system. The evidence supporting the role of glucocorticoids
in glutamine transport is indirect and rests on the fact
that the two glutamine utilizing systems are located in
separate cellular compartments. In the rat, glutamyl gluta¬
mine transferase is located in the cytoplasm and glutaminase
I is found in the mitochondrion. In the normal rat, approxi¬
mately 70% of ammonia production takes place in the cyto¬
plasm and 30% in the mitochondria (17). One mole of ammonia
13
is produced by the reaction catalyzed by GT, while two moles
of ammonia are derived from each mole of glutamine meta¬
bolized in the mitochondria (since glutamine undergoes both
deamidation and deamination). The ammonia/glutamine ratio
varies with the activity of the cytoplasmic and mitochondrial
enzymes, and is equal to 1.3 under normal circumstances (17).
If glutaminase I is inhibited the ratio falls and equals 1.0.
On the other hand, in metabolic acidemia, since 80% of
ammonia produced is derived from glutamine metabolized in
the mitochondrion the NH^/glutamine ratio rises to 1.5. In
normal rats, bilateral adrenalectomy resulted in a 30%
decrease in ammonia excretion despite the development of
metabolic acidemia. The NH^/glutamine ratio fell from 1.3
to 1.1 (50). This suggests that adrenalectomy inhibited
the mitochondrial metabolism of glutamine. Conversely,
the ingestion of triamcinolone increased ammonia production
by 3 fold in the face of a metabolic alkalemia (50). The
NH^/glutamine ratio increased from 1.3 to 1.5. This is con¬
sistent with an increase in the intramitochondrial metabolism
of glutamine. Since in vitro studies with kidneys from
adrenalectomized rats show the initial effect of triamcino¬
lone to be apparent within 15 minutes, it is unlikely that
the synthesis of additional molecules of glutaminase I was
responsible for the increase in ammoniagenesis. These re¬
sults are more consistent with an increase in mitochondrial
permeability to glutamine (5“0) .
14
D) Effect of glutaminase activity on NH^ production
Davies and Yudkin (21) were the first to demonstrate an
adaptive increase in glutaminase I activity during chronic
metabolic acidosis. However, the significance of this in¬
crease is unclear since during the first 24 hours of ammon¬
ium chloride administration, ammonium excretion and produc¬
tion increased without changes in glutaminase I activity
(51). Furthermore,the inhibition of protein synthesis by
actinomyosin D blocked the adaptive increase in renal gluta¬
minase I activity without altering the increase in ammonia
production (52). Interestingly, the increase in ammonia
production that occurs during acid loading and actinomyosin
D administration has been correlated with an increase in
the activity of the purine nucleotide cycle enzymes namely
adenylosuccinate synthetase adenylate deaminase and adenylo
succinase (53). It has thus been suggested that the pro¬
duction of ammonia from fumarate via the purine nucleotide
cycle may contribute significantly to the renal pool of
ammonia in acidosis. The net reaction for one turn of the
cycle is:
Aspartate + GTP + H20-^ Fumarate + NH^ + GDP + Pi
Experimental evidence demonstrating glutaminase II activity
(the liver enzyne) has not been forthcoming (54).
E) Effect of acidemia on NH^ production
Robinson (55) and Holmes (56) demonstrated that kidney
15
slices incubated in acid medium displayed increased ammonia
production and concluded that the intracellular acidemia
per se was responsible for the increase in ammoniagenesis.
However, other studies employing renal slices and mitochon¬
drial systems have shown the initial effect of acidemia to
be an inhibition of glutaminase I activity and a fall in
ammonia production (57,58). As the duration of acidemia
increased, the initial inhibition of glutaminase I was
overcome and ammonia production increased. Consequently,
acidemia per se does not appear to stimulate ammoniagenesis.
F) Effect of potassium hemeostasis on NH^ production
Potassium depletion has been shown to alter ammonia pro¬
duction in several ways: a) it stimulates glutamine I and
II, (59-62), b) it facilitates the entry of glutamine into
the mitochondrion, c) it increases the production of ammonia
from both glutamine and glutamate (63-66), and d) it in¬
hibits the conversion of glutamate to glutamine thereby
increasing the availability of ammonia for excretion (63-
66). Potassium loading on the other hand inhibits ammonia
production (67).
G) Effect of renal concentration of glutamate and NAD+/
NADH ratio
Glutamate inhibits the reaction catalyzed by glutaminase
During acidosis, the intracellular concentration of gluta-
J
16
mate falls and glutaminase I activity rises. Preuss has
proposed that the initial reduction in glutamate concentra¬
tion is caused by an increase in the redox state of pyridine
nucleotides (68-69). Direct and indirect measurements of
“t-
NAD /NADH and NADP /NADPH ratios showed them to be increased
in both acute and chronic acidemia. An increase in the
NAD+/NADH ratio will facilitate the conversion of glutamate
to a-ketoglutarate and thereby cause a fall in glutamate
concentration:
glutamate glutamate dehydrogenase
NAD NADH+
a-ketoglutarate + NH.
H) Effect of plasma ammonia concentration on NH^ production
Like glutamate}ammonia inhibits the deamidation of
glutamine (13). Pitts has shown that infusions of ammonium
chloride into the renal artery of dogs although increasing
urinary ammonia excretion, significantly inhibited renal
ammoniagenesis (70). Similar results were obtained with
infusions of N15 ammonium lactate (71). One cannot conclude
that the rise in arterial ammonia concentrations caused
the decline in ammonia production observed with ammonium
lactate infusion since it is known that lactate alone in¬
hibits renal ammoniagenesis in vivo and in vitro (71).
More importantly, the concentration of ammonia necessary
to achieve 50% inhibition of glutaminase I is two orders
of magnitude greater than those observed in vivo. Conse¬
quently, it is likely that this effect is not important
17
physiologically (13).
I) Effect of a-ketoglutarate concentration on NH^ produc¬
tion .
a-ketoglutarate which provides the carbon skeleton of
glutamine also inhibits glutaminase I and glutamic dehy¬
drogenase. Its cellular concentration decreases during
metabolic acidemia and increases in metabolic alkalemia
(14,15). Furthermore, infusions of a-ketoglutarate cause
a significant decrease in ammonia production (72). Although
glutamate concentration rises with a-ketoglutarate, the
rise is not sufficient to account for the entire decrease
in ammonia production. Consequently, a direct inhibition of
glutaminase I by a-ketoglutarate has been postulated (72).
J) Effect of renal gluconeogenesis on NH^ production
In metabolic acidemia, gluconeogenesis is stimulated
(73-75). This observation suggests that glucose formation
increases the utilization of glutamine and reduces tissue
glutamate concentration, thereby activating glutaminase I.
However, more recent experiments indicate that gluconeo¬
genesis and ammoniagenesis are dissociable. Churchill
et al showed that the inhibition of gluconeogenesis has
no effect on ammonia production in acidemia (76) and Pitts
documented that only 25% of the glutamine used by the
18
kidney is converted to glucose (77). It is now believed
that the increase in gluconeogenesis observed with meta¬
bolic acidemia is secondary to the rise in renal ammonia
production and is dependent upon the availability of
glutamate (78).
K) Effect of oxidizable substrate concentration on NH^
production
Ketone bodies, free fatty acids and lactate are all oxi¬
dized by the kidneys and changes in their availability have
been shown to alter renal ammonia production. Lemieux demon¬
strated a 50 to 60% fall in ammonia production and excretion
during ketone body infusions in ammonium chloride loaded
acidemic dogs (5). A simultaneous decrease in the renal
extraction of glutamine was also noted in these studies.
The inhibitory effect of ketones was independent of urinary
pH or acid base changes. Similar results have been obtained
with infusions of sodium citrate (79), sodium octanoate and
levarterenol (80). The inhibitory effect of ketone bodies
appears to be linked with their oxidation and can be elimina¬
ted by blocking the TCA cycle with fluoracetate (81). Lac¬
tate inhibits ammoniagenesis by kidney slices of acidemic
rats independent of changes in pH. (71). It is thus clear
that in animal experiments, both in vivo and in vitro, an
increase in substrate availability for oxidation by the
19
kidney can cause a decrease in the rate of ammoniagenesis.
In summary, in chronic metabolic acidemia, the following
alterations are thought to increase ammonia production:
a) decreased intracellular pH, b) decreased cellular po¬
tassium concentration, c) a decrease in the renal concentra¬
tion of glutamate, d) an increase in the NAD+/NADH ratio,
e) a fall in the concentration of ammonia and a-ketogluta-
rate, f) the stimulation of renal gluconeogenesis, g) an
increase in the availability of oxidizable substrate.
B. Ketone body metabolism
Regulation of peripheral uptake.
Ketone bodies are an important source of energy during
starvation (82) and long term exercise (83). The rate of
uptake and oxidation of beta-hydroxybutyric acid varies
directly with its plasma concentration (84,85) . During
prolonged starvation and diabetic ketoacidosis, however,
the peripheral uptake of ketones is depressed in spite of
their very high plasma levels (86,87). These observations
have stimulated interest in the role of insulin as a regu¬
lator of ketone metabolism. Acetoacetate uptake by the
liver and skeletal muscles is reduced in streptozotocin
induced diabetes in rats and is restored to normal with
insulin administration (88). Balasse has shown that ketone
body uptake by dog skeletal muscle is stimulated by insulin
20
(89) . In man, Felig and Sherwin have demonstrated that hy-
perinsulinemia stimulates beta-hydroxybutyrate utilization
(90) . Thus, both the plasma ketone and insulin concentra¬
tions appear to play important regulatory roles in modulat¬
ing ketone uptake by peripheral tissues.
Effects of ketone bodies on glucose metabolism.
Ketone bodies appear to inhibit glycolysis. In both
normal and diabetic subjects, infusions of Na 3-OHB while
not affecting the plasma concentration of insulin are
associated with a fall in plasma glucose (90). Randal has
shown that in the perfused rat heart and isolated rat
diaphragm, ketone bodies inhibit glucose metabolism at the
level of phosphofructokinase (91). On the other hand, in¬
hibition of glucose metabolism in the rat hindquarter could
not be demonstrated (91). Interestingly, lactate and pyru¬
vate oxidation and the activity of glutamate dehydrogenase
are markedly inhibited by ketone bodies (92). Gluconeo-
genesis is also inhibited by ketone bodies (93). Using
renal cortical slices from acidemic and normal dogs|Lemieux
showed that 3-OHB and acetoacetate in concentrations rang¬
ing from 0.5 to 5 mM induced a 20 to 30% decrease in glucose
production when either glutamine or glutamate was used as
substrate (93).
21
Effects of ketones on amino acid release.
Felig et al have demonstrated that in the postabsorptive
state and in prolonged starvation, intravenous fusion of
ketone bodies may inhibit the peripheral release of alanine
and results in a significant decrease in the plasma alanine
concentration (90,94). The urinary excretion of ammonia
was also reduced. In the diabetic rat, no effect of acetoace-
tate (1.5 mM) or Na 3-OHB (1.9 mM) on plasma alanine con¬
centration could be demonstrated in the absence of insulin
(95). As the plasma ketone concentration was increased
further, however, a significant decrease in plasma alanine
concentration occurred in the absence of insulin. Since
alanine is the principal amino acid extracted by the liver
for gluconeogenesis, (96) inhibition of its peripheral re¬
lease may explain the reduction in glucose production during
ketone infusion.
22
III. MATERIALS AND METHODS.
A. Infusion studies.
Seven healthy, (4 males, 3 females) normal weight volun¬
teers (body mass index = 20 ± 2), ranging in age from 19 to
36 years were studied. Over the three day period prior to
study, each subject consumed a weight maintaining diet con¬
taining at least 200 grams of carbohydrate per day and in¬
gested 0.1 g/kg body weight of ammonium chloride daily. The
ammonium chloride was administered in 4 divided doses at
six hour intervals, the last dose being administered at
twelve on the night prior to study. All the subjects tolera¬
ted the ammonium chloride well without nausea or vomiting.
Venous pH and bicarbonate concentration were measured prior
to starting ammonium chloride. Subjects remained fasting
after midnight on the evening prior to study. On awakening
at 7 am on the morning of study, they drank 600 ml of tap
water and reported to the Clinical Research Center at 8 am.
A water load of 20 ml/kg was administered from 8 to 8:30 am
and the volume of each subsequently voided urine was quan¬
titatively replaced to achieve a steady state water diuresis
of 9-12 ml/min.. Small polyethylene catheters were inserted
under local xylocaine anesthesis into each antecubital vein,
one for the infusion of all test substances and the other
for withdrawal of blood. Priming doses of insulin (40 mg/kg)
and PAH .(12 mg/kg) were administered and were immediately followed
23
by a continuous infusion of insulin and PAH at a rate of
1.11 ml/min. calculated to maintain plasma levels of 20 mg%
and 2 mg% respectively. The subjects remained supine through¬
out the study except to void. After a 60 minute equilibra¬
tion period, 3 consecutive 30 minute urine volumes were
collected. Subjects then received a 3 hour intravenous
infusion of sodium 3-hydroxubutyrate (n=7), sodium bicarbo¬
nate (n=3) or sodium lactate (n=4) as described below. Urine
was collected at 30 minute intervals throughout the study
period. Venous blood samples were drawn without the use
of a tourniquet at the end of each urine collection. All
blood losses were replacedquantitatively with intravenous
normal saline.
B. Infusion of test substances.
Sodium 3-hydroxybutyrate (Na 3-OHB) studies.
Sodium 3-hydroxybutyrate (Na 3-OHB was administered in¬
travenously as a priming dose ( lmmole/kg) over 20 minutes)
followed by a continuous infusion at a rate of 2.2 ml/min.
which delivered 0.01 mmole/kg min. of Na 3-OHB. The Na
3-OHB (Nutritional Biochemical Co., Cleveland, Ohio) was
prepared as a 40% solution in sterile water, passed through
a 22 pm filter apparatus (Millipore Corp., Bedford, Mass.)
and stored at 4°C until used. Each lot was tested for
pyrogenicity (97) and sterility prior to use. Sterile
pyrogen free water was added to the 40% solution to achieve
the desired ketone concentration. By varying the concentration
24
of the infusate, a continuous delivery rate of 2.2 ml/min.
was used in all patients.
Sodium bicarbonate (Na HC03) studies.
3 patients were restudied as previously described except
that they received an infusion of Na HC03. These subjects
served as control for volume expansion and for the degree
of alkalemia which was associated with Na 3-OHB infusion.
The rate of bicarbonate infusion varied from 0.52 to 0.85
meq/min. and was calculated to simulate the rise in plasma
bicarbonate observed during the infusion of Na 3-OHB. Insulin
and PAH were not infused in this or the subsequent study
with sodium lactate. Endogenous creatinine clearance was
used as an index of glomerular filtration rate (GFR).
Sodium lactate studies.
Four subjects were restudied with sodium lactate to
simulate the change in intracellular redox potential (NAD+/
NADH) that occurs during the conversion of 3-hydroxybutyrate
(B-OHB) to Acetoacetate (AcAc). The oxidation of lactate
to pyruvate causes a fall in the NAD+/NADH ratio. These
subjects were restudied in a similar manner to that previous¬
ly described except that they leceived an infusion of sodium
lactate, given as a prime (1 mmole/kg over 20 minutes) -
continuous unfusion (0.01 mmole/kg~min.).
25
The purpose and potential risks of the study were ex¬
plained to each subject and written, voluntary consent was
obtained prior to their participation.
C. Analytical methods.
Plasma and urine sodium and potassium concentrations
were determined by flame photometry (Flame Photometer 143
Instrumentation Lab. Inc., Watertown, Mass.). Urine and
plasma chloride concentrations were determined with a silver
electrode titrating chloridometer (Buchler-Cotlove, Chlorido
meter Buchler Instruments, Ft. Lee, N.J.). Plasma bicar¬
bonate concentration was determined by using a modification
of the Van Slyke method. Whole blood pH and PCC^ were
measured on a blood micro pH meter (Radiometer, Copenhagen).
Insulin concentrations in plasma and urine were quantified
by the method of Walser et al (98) adapted to the autoanalyz
er by Fuhr et al (99). Plasma and urine PAH were measured
by the method of Smith (100) using a Gilford spectrophoto¬
meter at 540 nm. Creatinine was determined by the method
of Folin and Wu (101) adapted to the Technicon Autoanalizer.
Plasma and urine B-OHB and acetoacetate concentrations were
measured according to the method of Williamson and Krebs
(102). Plasma lactate was determined using an enzymatic
assay (103). Plasma glucose was measured by the glucose
oxidase method (104) on a Beckman Glucose analyser (Beckman
Instrument Inc. Science Essentials Co., Mountainside, N.J.).
26
Plasma free fatty acids were measured using the modified
Doles' Titrimetric method (105). Plasma glutamate was deter¬
mined by enzymatic reaction with glutamate dehydrogenase
(GDmp4) (L glutamate, NADP oxydoreductase deaminating, EC
1.4.13) (106). Plasma glutamine concentration was quantified
by enzymatic hydrolysis with purified glutaminase (L gluta¬
mine aminohydrolase E.C. 3512), followed by spectrophoto-
metric detection of glutamate at 340 nm(106). The methods
used for the determination of plasma immunoreactive insulin
and glucagon andplasma acidic and neutral amino acids have
been previously described (107) . Urine was collected under
oil and analyzed for ammonium, titratable acidity and pH on
the day of study. Urinary pH was measured wtih a glass elec¬
trode pH meter (PHm 71 Radiometer, Copenhagen, Denmark).
Urinary ammonium and titratable acidity were determined
using the microdiffusion method of Conway (108) and the
method of Peters and Van Slyke (109) respectively.
D. Calculations.
For the statistical analysis of the data, observations
from the 3 control periods before the administration of
test substances were averaged and compared to the values ob¬
tained following the infusions (paired t-test) , (110) . Each
subject thereby served as his own control. Differences be¬
tween the hydroxybutyrate, lactate and bicarbonate groups
27
were compared using the unpaired t-test. Absolute urinary
excretion of electrolytes were determined using the formula:
Excretion = Urinary concentration urine flow (pmol/min.).
The clearances of creatinine, PAH and Insulin were calculated
using the formula: Cx = Ux.V/Px. All values represent mean
+ SEM.
28
IV. RESULTS
1. NA g-OHB infusion studies
A. Chronic ammonium chloride loading. (Table 1): After 3
days of ammonium chloride ingestion, the venous pH fell from
7.36 ± 0.02 to 7.31 ± 0.12 (p <0.01) and the plasma bicar¬
bonate concentration decreased from 25 + 1 to 20 + 1 meq/L
(p <0.01). There were no significant changes in body weight
and plasma concentration of sodium, chloride and potassium.
B. Infusion of Na g-OHB: During the infusion of Na 3-OHB,
the rise in urinary 3-OHB excretion paralleled the increase
in plasma ketone concentration. Within 30 minutes, plasma
ketone concentration increased from 81 + 12 to 530 + 20
yM (p <0.001) and then gradually declined to 193 + 29 yM (p <0.001)
by the third hour. Urinary 3-OHB excretion rose from 0.7 ±
0.008 to 136 + 48.4 ymol./min (p <0.002) at 30 minutes
and then gradually fell to a nadir of 23.7 ± 6 ymol./min (p <0.01)
at 120 minutes, (Table 2 and 3, Figure 4B). The pattern of
change in plasma acetoacetate concentration was similar to
that of B-OHB. Throughout the study, urine flow rate and
creatinine, Inulin and PAH clearances remained unchanged
from baseline (Table 2). Baseline urine pH (5.63 + 0.06)
fell to 5.34 ± 0.056 (p <0.02) at 30 minutes and then gradu¬
ally rose reaching a value of 5.97 + 0.008 (p < 0.02) at the
end of 3 hours. Urinary ammonium excretion fell by 26% ± 2
29
(p <0.01) within 30 minutes after starting the infusion of
Na 3-OHB, despite a significant fall in urine pH (Table 2).
Thereafter, ammonia excretion continued to decrease reaching
a nadir of 54 + 5.5 yeq/min. (p <0.002) . This represented
a 43% decrease in ammonia excretion at 120 minutes. During
the last hour of the study ammonium excretion remained
suppressed (Table 2, Figure 5). Over the entire study period,
the mean decrease in ammonium excretion was 35% less than
the basal excretion rate (p <0.001). The excretion of tit-
ratable acid followed a slightly different pattern. No sig¬
nificant change from baseline (41 + 3 yeq/min.) was observed
at 30 minutes but thereafter it gradually fell reaching a
level of 28.3 + 1.7 yeq/min. (p <0.02) at 3 hours (Table 2).
Urinary sodium excretion (U, V) fell from a baseline value Na
of 221 + 18 to 124 + 11 yeq/min. (p<0.01) (Table 4, Figure
6) without a change in plasma sodium concentration (P„T ) or
Inulin clearance (C ). Consequently, the fractional ex¬
cretion of sodium (C /C ), fell proportionately to the de¬
crease in U^ V. The pattern and the magnitude of change in
UCiV, C and C^/C paralleled that of sodium. The plasma
chloride concentration decreased from 103.44 + 0.9 to 98.8 +
0.65 meq/L (p < 0.01), (Table 4). The plasma potassium con¬
centration fell from 4.18 + 0.09 to 3.55 + 0.05 meq/L
(p < 0.001) at 3 hours (Table 5). Urinary potassium excretion
(U V) remained unchanged during the first hour but thereafter
fell progressively, reaching a value 31% below baseline values
30
at 3 hours. Since urinary potassium excretion fell in con¬
cert with the decrease in P , there was no significant change lx
in CK and CK/CIN‘
The infusion of Na 3-OHB was accompanied by a gradual
rise in venous pH from 7.31 + .012 to a peak of 7.38 + 0.01
(p <0.01) and bicarbonate concentration also increased from
19.4 + 1 to 24.8 + 1.1 meq/L (p <0.02), (Table 6). The plasma
concentration of glucose, insulin, glucagon and free fatty
acids remained unchanged (Table 7). Plasma amino acid con¬
centration remained the same for that of alanine which de¬
creased by 20% at 150 minutes. Glutamine and glutamate con¬
centrations were unchanged. (Table 8).
2. NaHCO ^ infusion studies.
A. Chronic ammonium chloride loading: Following the 3 day
period of ammonium chloride administration, venous pH and
plasma bicarbonate concentration decreased from 7.36 + 0.03
to 7.30 + 0.004 (p <0.05) and from 25.4 + 0.8 to 19.4 + 1.1
meq/L (p <0.01) respectively. There were no significant
changes in body weight and plasma sodium, chloride and
potassium concentration (Table 1).
B. Infusion of NaHCO^: Following NaHCO^ infusion venous pH
rose from 7130 + 0.01 to 7.38 + 0.01 (p < 0.01) and plasma
bicarbonate concentration from 19.5 + 0.3 to 26 + 0.2 yeq/L
p <0.002). These increases were similar to those observed
31
in the Na 3-OHB infusion studies, (Table 6 and 11). Urine
flow and creatinine clearance were unchanged from baseline.
Urine pH gradually increased from 5.64 + 0.06 to 6.1 + 0.06
(p <0.01) at 180 minutes (Table 9). The rise in urine pH
was similar to that observed with Na 3-OHB. Ammonium ex¬
cretion was unchanged from baseline, 92+8 yeq/min, at 30
minutes, compared to a 26% decrease (p <0.01) observed follow¬
ing Na 3-OHB infusion. At 120 minutes following NaHCO^
infusion, ammonium excretion decreased slightly, 84+5 yeq/
min.. This decrease was not statistically significant-.
At 180 minutes, however, ammonium excretion fell further to
74+5 yeq/min. (p <0.05) (Table 9). The decline in ammonium
excretion was only half as great as that observed with Na 3-
OHB infusion (p <0.01). Over the entire study period, the
mean fall in ammonium excretion was 8% of the baseline value,
compared to a 35%- decline observed following Na 3-OHB (p <
0.002). No significant change in titratable acid excretion
occurred. U„T V decreased from a control value of 210 + 16 to
112 + 11 yeq/min at 180 minutes (p <0.01) without any change
in plasma sodium concentration (Table 10). Sodium clearance
declined from a baseline value of 1.6 + 0.12 to 0.9 + 0.1
ml/min (p <0.01). (Table 19, Figure 7). Plasma chloride
concentration fell from 104.0 + 0.2 to 99.0 + 0.4 meq/L
(p <0.02) and this was paralleled by a decrease in chloride
excretion from 222 + 36 to 163 + 19 yeq/min (p <0.05) . The
32
clearance of chloride decreased similarly from 2.1 + 0.2 to
1.64 + 0.1 ml/min (p <0.05), (Table 10). U V and C did not
change significantly even though the plasma potassium concen¬
tration decreased from 4.54 + 0.03 to 3.95 + 0.1 3ieq/L (p <
0.05). There was no change in plasma ketone glucose, insulin,
glucagon, free fatty acids and amino acid concentration (Table
12,13).
3. Na Lactate infusion studies.
A. Chronic ammonium chloride loading: During the three day
ammonium chloride loading, venous pH fell from 7.36 ± 0.01
to 7.30 + 0.01 (p <0.05) and the plasma bicarbonate concen¬
tration decreased from 25.4 + 0.8 to 18.3 + 1.5 pleq/L (p <
0.01). There was a small and statistically insignificant rise
in plasma chloride and potassium concentration. No significant
changes in plasma sodium concentration and body weight were
observed (Table 1) .
B. Na Lactate infusion: Plasma lactate concentration rose
from 6.9 + 0.5 to 13.7 + 1 mM (p < 0.002) within 30 minutes
and reached a plateau of approximately 10 mM by 60-90
minutes. No change in plasma ketone concentration was observed
(Table 14, Figure 6A). Venous pH increased from 7.30 + 0.01
to 7.38 + 0.01 (p <0.01) and plasma bicarbonate concentration,
from 18.3 ± 1.5 to 25.8 ± 0.8 meq/L (p <0.01) (Table 15).
Creatinine clearance and urine flow were unchanged from base-
33
line. Urinary pH fell from 5.12 + 0.018 to 5.02 + 0.025 at
30 minutes and then gradually increased reaching a value of
6.03 + 0.16 (p <0.01) at 180 minutes (Table 16). Urinary
ammonium excretion decreased by 10% at 30 minutes and pro¬
gressively fell thereafter reaching a value which was 40%
below baseline at 150-180 minutes (p <0.01) (Table 16, Figure
8). The mean decrease in ammonium excretion during the 3 hour
study was 35% (p <0.01). The change in ammonium excretion
was similar to that observed following Na B-OHB infusion.
Urinary sodium excretion and sodium clearance fell signifi¬
cantly by 30 minutes and reached a plateau value, 60% below
baseline, at 120 minutes (p <0.001), Figure 7. No change
in plasma sodium concentration occurred (Table 16). Urinary
chloride excretion and chloride clearance paralleled those
of sodium. Plasma chloride concentration fell significantly
from 102 ± 1 to 98 + 1 meq/L (p <0.05) . Urinary potassium
excretion and potassium clearance fell by 55% (p <0.05) and
46% (p <0.05) at 150 minutes, and this was associated with
a decrease in plasma potassium concentration from 4.23 + 0.2
to 3.5 + 0.15 meq/L (p <0.05), (Table 17). There was no
change in plasma glucose, insulin, glucagon free fatty acids
and amino acid concentration, (Table 18,19).
34
V. DISCUSSION
The present study demonstrates that the intravenous
infusion of Na f-OHB or lactate to acidemic subjects acutely
inhibits urinary ammonium excretion. The maximum increase
in plasma ketone concentration, 530 p M was modest and com¬
parable to levels seen in mild fasting and diabetic ketosis
(111). A similar effect of ketone infusion on urinary
ammonium excretion in acidemic dogs has been reported by
Lemieux et al (5). They showed that an increase in total
plasma ketone concentration to 4.50 mM led to an inhibition
of ammonia production and excretion which was associated
with a decrease in glutamine uptake.
The factors known to affect renal ammonium excretion
and production are listed in table 20, and are shown schematic¬
ally in figure 10. The sources of excreted ammonium are
shown in figure 3.
In the present study, blood and urine pH rose following
Na 3-OHB infusion. Although those changes could potentially
explain the decrease in ammonium excretion, several observa¬
tions suggest that other mechanisms must also be involved.
The initial (0-30 min) effect of Na 3-OHB infusion was to de¬
crease urine pH (p<0.002), which should have increased ammon¬
ium excretion since the lower urine pH would trap more
ammonia in the ionized form (26). Instead ammonium excretion
fell by 27% (p <0.01) during the first 30 minutes. Addition-
35
ally, neither blood pH nor plasma bicarbonate concentration
changed significantly during this time period. To further
examine the potential contribution of changes in blood and
urine pH to the decrease in ammonium excretion, 3 subjects
were restudied with an infusion of sodium bicarbonate de¬
signed to increase the plasma bicarbonate concentration and
blood and urine pH to levels similar to those observed
during Na 3-OHB infusion. Despite a similar or greater plas
ma bicarbonate concentration (23 meq/L with Na 3-OHB vs 26
with NaHCO^)/ blood pH (7.36 with Na 3-OHB vs 7.38 with
NaHCO^) and urine pH (5.97 with Na 3-OHB vs 6.10 with
NaHCO^) the mean decline in urinary ammonium excretion was
3.5 times greater with Na 3-OHB than with NaHCO^. Thus,
it appears likely that factors other than the rise in plasma
bicarbonate concentration and blood and urine pH must have
contributed to the decline in ammonia excretion following
Na 3-OHB infusion.
Since urine flow did not change during our experiments
an increase in back diffusion of ammonia (37) or a change
in the concentration gradient from the tubular lumen to the
renal tubular cell cannot explain the observed decrease in
ammonia excretion. Since there is no evidence to suggest
that ketones inhibit hepatic ammonia production, it is
unlikely that decreased arterial ammonium concentration can
explain the decline in ammonium excretion observed during
36
Na 3-OHB infusion. Since RBF and GFR were unchanged and
since plasma levels of glutamine were unchanged, it is un¬
likely that decreased substrate delivery can account for
the fall in ammonia excretion.
Lemieux et al have shown previously that ketones in¬
hibit renal ammoniagenesis both in vivo and in vitro (5,87) .
Although the renal extraction of glutamine was significantly
depressed in the in vivo experiments, the uptake of gluta¬
mine by the renal tubular cell from the renal tubular lumen
(5) was not affected. These authors postulated that the meta¬
bolism of ketone bodies by the renal tubular cell caused
intracellular changes that inhibited ammonia production. In
vitro studies suggest that ketone bodies inhibit glutamine
metabolism through a direct metabolic effect linked with
ketone oxidation in the tricarboxylic acid cycle (81). In
renal cortex slices from acidotic dogs, ketones inhibit
glutamine uptake, ammoniagenesis and gluconeogenesis and
these effects can be completely reversed by the addition of
fluoracetate which blocks the TCA cycle. Fluoracetate is
known to act by inhibiting the enzyme aconitase which converts
citrate to isocitrate (81). However, although it seems that
oxidation of ketones via the TCA cycle results in the in¬
hibition of renal ammoniagenesis, the exact mechanism by
which this occurs is not clear at present.
Ketones could interfere with ammonia production in two
ways: first, by increasing the intracellular a-ketoglutarate
37
level or secondly by decreasing the NAD+/NAL>H ratio in the
renal tubular cell. Ketone bodies are first oxidized to
Acetyl CoA which subsequently enters the TCA cycle. Citrate
synthetase then catalizes the formation of citrate from
Acetyl CoA and oxaloacetate (Figure 1). Citrate is in turn
converted to a-ketoglutarate which plays an important role
in ammonia production. Increased levels of a-ketoglutarate
inhibit both the transport and the deamidation of glutamine
by renal mitochondria (14,15). The intravenous infusion of
a-ketoglutarate into chronically acidotic dogs decreases the
renal production of ammonia and increases the amount of
glutamate and a-ketoglutarate that is released in the renal
vein (72). It is thought that an increase in cellular a-
ketoglutarate concentration inhibits the deamination of
glutamate. Since glutamate is an inhibitor of glutaminase I,
a rise in its concentration will inhibit the deamidation of
glutamine (Figure 1). Although it is clear that an increase
in a-ketoglutarate concentration can result in a fall in
ammonia production, it has not been proven that the oxidation
of ketone bodies causes a significant rise in a-ketoglutarate
concentration. Indeed, when Lemieux incubated renal cortical
slices from acidemic dogs with ketone bodies he observed a
significant decline in ammonia production but could not de¬
tect any change in a-ketoglutarate concentration (87).
The second mechanism by which hyperketonemia may inhibit
38
ammonia production is related to a decrease in the NAD /NADH
ratio which has been postulated to occur during ketone oxi¬
dation :
3-OHB NAEuNADH_^ Acetoacetate (AcAc)
As B-OHB is converted to AcAc the concentration of NAD de¬
creases while that of NADH increases. This results in a
decline in the NAD+/NADH ratio. Both Pitts (27) and Preuss
(68/69)have underscored the theoretical importance of the
redox state of nicotinamide dinucleotide in the control of
ammonia production. Preuss has demonstrated an increase in
the NAD+/NADH ratio in both acute and chronic acidemia.
Glutamate dehydrogenase which catalyzes the deamination of
glutamate to a-ketoglutarate is in equilibrium with the
B-OHB dehydrogenase system and is therefore influenced by
the NAD+/NADH ratio (16). A rise in the ratio inhibits the
deamination of glutamate leading to the accumulation of
glutamate which can in turn depress glutaminase I activity.
This will decrease the rate at which glutamine is deaminated
and will result in decreased ammonia production (68,69).
To determine whether an alteration in the NAD+/NADH
ratio might play some role in the fall of ammonia production
observed following hyperketonemia, we infused Na lactate
into chronically acidemic subjects to stimulate the conversion
of NAD /NADH. Lactate is converted to pyruvate in the cytosol
and NADH is generated from NAD+ in the process:
Lactate NAD+ NADH ^ Pyruvate
39
Following lactate infusion, urinary ammonium excretion de¬
clined by 35% (p <0.01) and this decline was similar to
that observed with Na 3-OHB. Although Lemieux et al could
not demonstrate a fall in ammonia production in acidemic
dogs during ketone body infusion, other investigators have
previously reported the inhibition of ammonia production by
lactate both in vivo (71) and in vitro (111,112) . Thus the
results observed with lactate infusion are consistent with
the possibility that the decrease in the NAD-/NADH ratio
is responsible for the decline in ammonia excretion.
Potassium homeostasis is thought to be closely related
to ammonia production. Both in vivo and in vitro studies
have demonstrated thatrpotassium depletion stimulated ammonia
production (63-66). Several mechanisms have been shown to
play a role in the increase in ammonia production following
potassium depletion: 1) Stimulation of glutaminase I activity
(59-62), 2) Stimulation of glutaminase II activity (59-62),
3) inhibition of glutamine synthetase activity and of the
conversion of glutamate to glutamine (66). In contrast,
potassium loading has been shown to depress ammonia pro¬
duction (67). This inhibitory effect of potassium on
ammoniagenesis has not been well studied. The only available
evidence suggests that potassium loading does not affect
glutaminase I activity (113) .
Plasma potassium concentration decreased'significantly
following both Na (30OHB (p <0.001) and Na lactate (p <0.002)
while urinary potassium excretion decreased or remained the
40
same. The decline in plasma potassium concentration is most
likely related to the increases in blood pH and plasma bi¬
carbonate concentration with a subsequent shift of hydrogen
ion out of cells in exchange for potassium (114-116) . Measure¬
ments of the potassium transport pool in distal tubular cells
of rats using isotopic potassium have shown that alkalosis
increases the intracellular pool size of potassium (117) .
Consequently, in the present study, it is likely that cellu¬
lar potassium concentration increased during b oth Na 3-OHB
and Na lactate infusion. This could represent a situation
similar to that observed following potassium loading. Thus,
it is possible that the decline in urinary ammonium excretion
was in part due to an increase in intracellular potassium
concentration. This explanation is unlikely for several rea¬
sons :
1) ammonium excretion falls before any change in plas¬
ma potassium concentration occurs. 2) Tannen has shown that
acute changes in intracellular or extracellular potassium
concentration or in the transcellular potassium gradient do
not influence ammoniagenesis (118). 3) In the present study
although NaHCO infusion resulted in a significant rise in
blood pH and a decrease in plasma potassium concentration,
the decline in urinary ammonium excretion was significantly
less than observed with Na 3-OHB or Na lactate.
Several studies have suggested that ammonia production
is linked to gluconeogenesis (73-75). Since ketones are
41
known to inhibit renal gluconeogenesis (73,119) it is possi¬
ble that the decrease in ammonium excretion observed follow¬
ing 3-OHB in our studies was related to its inhibitory effect
on glucose production. Using renal cortical slices from
normal and acidotic dogs, Lemieux has shown the addition
of 3-OHB or AcAc (0.5-5 mM/L) leads to a 20% decrease in
glucose production and a 50% decline in ammonia production
(93). These authors postulated that decreased glucose pro¬
duction led to increased tissue glutamate and decreased
glutaminase I activity and ammonia production (Figure 1).
Conversely, it has been argued that enhanced gluconeogenesis
would stimulate ammonia production by reducing tissue gluta¬
mate and increasing glutaminase I activity. However, not
all of the available evidence supports such a strong link
between ammoniagenesis and glucose production. First,
Churchill and Malvin were able to clearly dissociate glucose
and ammonia production in vivo and in vitro (76). Second,
although lactate increases glucose production it depresses
ammoniagenesis (111).
In the present study, Na 3-OHB infusion caused a 20%
decrease in plasma alanine concentration. A similar fall in
plasma alanine level following ketone infusion has been re¬
ported by Felig in subjects fasted overnight as well as
following prolonged starvation (94). These authors suggested
that ketone bodies may inhibit muscle efflux of alanine or
possibly enhance hepatic alanine uptake. Studies by Pitts
42
have shown that about 5% of ammonia production is derived
from alanine. Since plasma alanine levels dropped by only
20% it is difficult to see how decreased alanine availability
could account for a 35% decrease in ammonia production follow¬
ing Na B-OHB infusion. Although Felig has suggested that
decreased muscle efflux or increased hepatic uptake of
alanine may be responsible for 3-OHB induced hypoalaninemia,
another possibility needs to be considered. Experiments in
14 which carboxyl C labeled pyruvate was infused into the
renal artery of acidemic dogs have shown that alanine is
produced by the kidney by the transamination of pyruvate
with the amino nitrogen of glutamine, either directly or
after the conversion of glutamine to glutamate (32,34).
Since substantial amounts of alanine are thought to be pro¬
duced by the kidney and since alanine production is depen¬
dent on renal ammonia production, ketone induced hypo-
alaninemia might result from the inhibition of renal ammonia-
genesis by ketone bodies and not vice versa.
In all 3 studies ( B-OHB, HCO^, lactate) urinary
sodium and chloride excretion and clearance declined without
any change in plasma sodium or potassium concentration.
This inhibition of sodium and chloride excretion was ob¬
served wtihin 30 minutes and was progressive throughout
the 3 hour study period. The fall in sodium and chloride
excretion occurred despite expansion of the extracellulary
volume (ECV). Indeed, ECV expansion would be expected to
43
increase both sodium and chloride excretion (120-124). Pre¬
sumably such ECV increase would have depressed aldosterone
levels which in turn would have led to an increase in sodium
and chloride excretion. The infusion of a poorly reabsorb-
able anion such as ketones would also be expected to enhance
sodium excretion (125). What then is responsible for the
observed decline in sodium and chloride excretion? It is
well known that metabolic acidemia inhibits renal tubular
sodium reabsorption (126-128) and results in a natriuresis.
Alkalemia does the opposite. Thus, the most likely explana¬
tion for the decrease in urinary sodium excretion is a change
in acid base status from chronic metabolic acidemia to acute
metabolic alkalosis. Such an explanation would account for
the fall in urinary sodium and chloride observed with Na
lactate, NaHCO^ and Na B-OHB.
Urinary potassium excretion decreased with lactate
and 6-OHB infusions. This finding was somewhat unexpected
since 1) both infusions were accompanied by a rise in plasma
bicarbonate and venous pH which should have resulted in an
increase in intracellular potassium concentration and a
subsequent rise in potassium secretion (117, 2) urinary
B-OHB and bicarbonate ion excretion increased significantly
with Na 3-OHB infusion; this should have caused a decrease
in the transepithelial potential difference and favored the
passive diffusion of potassium out of the cell into the
tubular lumen. A possible explanation for the observed
44
decrease in potassium excretion is related to the fact that
urinary sodium excretion fell with B-OHB and lactate infusion.
Indeed, a direct relationship has been observed between
potassium secretion and distal sodium delivery and reabsorp¬
tion (129): when sodium delivery is diminished, potassium
secretion is reduced. However, this explanation cannot
totally account for the decline in potassium excretion!
1) Recent evidence suggests that potassium secretion is
dependent on luminal flow rate and not on luminal Na con¬
centration (130). 2) NaHCO^ infusion results in a similar
decrease in sodium excretion while slightly increasing
potassium excretion.
45
Summary
This work was undertaken to determine the effects of
sodium beta-hydroxybutyrate on urinary ammonium excretion in
chronically acidemic human subjects. The following conclus¬
ions can be drawn from the above experiments:
1) Na3-0HB infusion significantly depresses urinary
ammonium excretion in chronically acidemic human subjects.
2) This effect of Na 3-OHB is unrelated to changes
in urine pH or in systemic acid base balance.
3) A fall in NAD+/NADH ratio may explain the inhibi¬
tory effect of Na 3-OHB on urinary ammonium excretion.
4) Na 3-OHB infusion caused a 20% decrease in plasma
alanine concentration.
5) 3-OHB, lactate and bicarbonate is accompanied by
a fall in urinary sodium excretion.
6) A decrease in potassium excretion is observed
with the infusion of 3-OHB and lactate.
Other experiments which would help determine the
precise nature of the inhibitory effect of 3-OHB on ammonia
production include: 1) infusing NAD+ and Na3-OHB or 3-hy-
droxy butryic acid in order to maintain the subjects in
metabolic acidemia. 2) Infusing acetoacetate, thereby
bypassing the following reaction:
3-OHB NAD+ NADH
Acetoacetate
+ 3) Directly measuring the NAD /NADH ratio, in
in vitro studies.
TA
BL
E
1.
Effect
of
a
3
day p
erio
d
of
am
moniu
m chloride
loading
on
pla
sm
a
electro
ly
tes,
ven
ou
s
blo
od
gases
an
d
bo
dy w
eig
ht.
46
MD LO o o oo r~-
V r—\ i—1 CM CM iO cO P 4* 1 + +1 +1 +1 + a O o co O
•h a • • • • • • CD P !—i o i—i o CO cO 2 o CO CO CO cO
-X ~
r~ o co 1-1 I> CM
a • • • • • • ro \ o rH o l-\ O i—1 o g1 + i + 1 +1 + 1 + 1 + 1 U (D in o m LO i—1
X £ CM CM CM i—1 CM CM
Q O O ro a i • cO o O CO
a D ro 1—1 • • • •
CM X CM CM ro o O £ + 1 +1 + 1 + 1 + 1 +1 U £ CM r—^ i—l r- CM ro
a ■cT 'M1 ro V1
CM i—1 i—1 i—i i—i i—l o O O o o o
+1 -H + 1 +1 +1 + X iO rH cO o [> o a ro ro ro ro ro ro
o !> r- [> r-
n-
o ro a + i o \ 00 U O' • X CD
£ CM
00
a o \ + 1
iH <2 r—1 U CD o
E r—1
c s uo
• CO CM CO < • CD X a + 1
-H a \ V1 p c 1—1 P a cd •
4-> £ co
c H 0 +1
•H a in CO \ • 0 rO 2 r-
+1 2 (D ro
t £ i—1 ■H
a a X o o
1 a oa 2
ra CD 2 g
0 4-1
• CD < a
o
i—i + UO
• c+ 1—1
o
rH
+1 ro O i—1
Oc O
+1 00 I—1
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rH + . ro CO
• (D ■H
ro P i—! a
a 10
a u ro
O 2 U 2 2
r0 g 2 CD + 44 •
<c 2
ro -
• ro i—I .
+ 1 4-1 CM LO
• • LO OJ i—i
CTr CM
O O + 1 +i
CM O O i—1 H
ro , 00 O CO 1—i • CD
• O ■H +1 + 1 P o P
in + . • CO
•M1 £ 0
i—1 i—i ■H + 1 + CO o O p
• • a co ro P ro ro •H H i—1
CD a
i—1 a co u u a H ■v o 2 2 ro 2 2 r—!
CD Ip U g 2 0 CD
44 a CD a • 2 < u
— i—I O LD O
i—I i—I o + 1+1 V ro ro
. . g in oo CM H -x
oo ro in • • o
O 1—I • + 1 + ! O O CM V O O a i—! i—[
O o c\j ro .
. o + i+i s O ro M cm cm 2
^ +1
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+ i +1 o o
co ro ro f—! i—i
a a o u "vT "sf I—) 2 a: o xx >
CD U L 0 (D a a 0) a 2 <C
ues
are
mean
G CM CO cD LD - Dr
■H • • • • LD •
g CO 1—1 i—1 !-1 « co 1-1
\ -H +1 -H - + 1 +1 • + 1 > • CM ID CM CM i—i CO
< p1 • • • • — • +1 • 0
T3 ft w i—1 o i—i o CO ft
g D _ ZL op “sf ^p CO CO CO CM •H
0 * -x- -X -X -X -X CO 0 II 0 . •M1 oc *LD -X LD * lD -X ID 1—1 G > 0
G G • • • • • • • ft •H PQ -H ■H oP ro OP ^P LD op cD 0 PQ g £0 i—i G 6 -H +1 +1 + 1 + 1 + 1 +1 ft 0 CM p 0 > \ cD H ac CO O 0 O Ift C •H co ft • . . • • • • ft 1 0 CQ0
■H 4-> X ft LD O ^P rH OP ac O'! 0 ca G D ft
0 2 ZL O CD cD LD LD LD G 0 0
G D 0 0 ft •v G 0 0 • > ft II ft ft
O X G r - -X 0 ftft •H
G 0 -H 00 00 CO o -X -X -X • ft
rd > g o h CM 1—1 cD 00 0 0 0.0 G PQ PQ \ • +l +1 +1 + 1 +1 + 1 ft •H o G II id X X 0 -H CM LD cD o [> ft 0 •H
> 0 o O f—f o » • • • • • 0 •- G H 1 1 0 r- CD 00 cD "sp 00 CM 0 G 0 P < 0 ca ca g > CO cD CO CM CM CO ft P a ft
D ZL o rH r G •G c II ft 0 ft! rvD ac l> CO 0 ft 0 f-r hM c G cD LD o 00 rH CO 0 G ft-
•H 0 O O o i—f o i—1 o 0 G 0 ft G G • . • • • • • G 0 0 0
■H "d EC +1 -H -H + 1 + 1 + 1 +1 ft ft 1-1 • -> -H
4-> •H ft CO '-P ID CO co CO o 0 0 a 0 -p CM rd 0 cD CO •M1 cD o O'! ac G £ a 0 O
0 0 ft • 1 . • • • • c G G O G LD LD LD LD LD LD LD ft r-1 -H 0 0 • U 0 ■ 0 ft i—1 G X o
i—1 G ■H 0 p 0 0 V
rQ X ■H r- r—1 OP O 'cP O LD G 0 G 0 ft > 0 < g CO lD ■M1 CO cD CO 0 ft H H 0 0 ft ft \ + 1 +1 -H +1 + 1 + 1 -H ft 0 0 •H -X
r~H 0 u 1—1 co O o CO a> ac O II G -X M-l G g st1 CO CD H1 H CD H ft 0 0
ft ID LD LD LD lD LD LD 0 G • G g • V
0 ■H G ■H ftG g r—l G ft ft G mg 0 o
■H G P O •H • G • 0 ft ft II o P 0 G 0 < 0
> V
-H -H cD cD •CP CO CD LD ft O 0 G G G g +1 +1 -H +1 +1 + 1 +1 0 0 G CO ft 0 0 H \ l> 1—1 cD CD CD r—1 ft C 0 a ft
g U 1-1 O i—1 O O O i—1 O Eh •H g 2 -X
G 0 g i—1 rH i—1 rH i—1 i—I, r—1 0 ■H ii ft 0 • ft -p
*H •s £ ft G «r • -
0 • • 0 •p a G CM P X ft 0 o 0 o
•ft ft • 0 •H •
G • ft ft ft ft o ■H 0 G G 0 0 0 V
c t +1 0 ft u G pq ■H G g ID CD CD CD 00 g G G U ft ft G 0 \ +1 +1 +1 +1 + 1 + 1 + 1 0 0 0 0 X o P U f—\ •O' CO lD CO cD CM O G G ft G 0 =
i g CM CM CO CO CM "sf 0 P 0 0 CO. i—1 i—i 1—1 i—1 rH rH *—1 0 0 0 >1
0 g 0 0 i-1 G rd 0 0 nj U 0 LD 2 G > 0 g £ G • O
0 0 G 0 •H G • m G !-S • cn r- o O CD CO co 0 0 G ft G 0 o 0 0 ft c • • . • • f • G 0 0 P ■ ft
0 ■H o o 1—1 i—1 O o o 0 •H -H G ft M -p i—1 0 g +1 + 1 -H -H +1 +1 +1 0 r—i 0 PQ 0 V
o 0 G \ CO o ac CO LD CM o P 0 P ft ft G 0 •H i—1 . • . • • • . i—! 0 ft ft O U ft
M—i X G g CM CO CM CM i—! CM 1—l n3 0 G •H 1 X ft < ft i—1 rH i—1 rH i—1 i—1 1—1 > ft •H ft ca 0 -
W a
CM 0 H 0 0 O o o
w ft G o o CM LD 00 ft 0 p ft O cD CJc i—1 i—1 i—i PQ g c G co 1 1 1 1 i C •H ■H 0 1 O o O o o ft ft 2 U o CO cD O'! CM LD
i—1 !-1
Effect
of
Na
3-O
HB
infusion
on
blo
od k
eto
ne
co
ncen
tration.
48
x x -x
o CM CM
i—1 + 1 s + O 3- 1—1 CO
CO IT)
-X ,—„ -X
o -X
< CD CO
o S + ft1
< a 20 cD —' CM CO
I—I
-X
-X
CQ -X
X o O CO CO
1 s + + 1 CQ. CO '—' LD co
CO
CO 0 i—1 O O
W ft g ft 0) 0 ft o CQ 6 G G CO < "H -H O 1 Eh Eh U o
■X X X X X
X X X X X
X X X X X
o CO CO o CM 1—1 i—t CM
+1 + 1 + 1 + 1 + i r- CM CD CO
CM cD CD CM IT) CO CM CM CO CO
X X X
X X X X X
X X X X X
o X 1—I X CO
1—1 oo r—1 CO 1—1
+ 1 +1 +1 +1 +1 CO CO 'sT CO
CO 00 O' CD r-
X X X X X
X X X X X
X X X X X
o CD CO CM CO CO I—1 1—I CM CM
+1 ft1 +1 +1 +1 CO CM CO CO
CO r- CO IT) CO
CM 1—1 1—1 CM 1—1
o o o UO CO
o o CM 1—1 1—1
cD
1
CO
1
1—1
1
1
o 1
o o o o CM uo CO cD CO 1—1 1—l
0 •V
0 ft ft c
4-1 G 0 0
G •H G •H
CD G kO ft
0 G ft 0
CD b G G
G ft ft
a b >1 G
CD CD X 0 G G 0 0
*H G c
b ffl b 0
0 ft 0
-H ft ft G 0 1 0
CD CQ ft ft co 0
ft 0 ft c 1—1 G £ 0 0
0 CD CO 0 •H
G £ 0 0 ft
4-) 0 1—1 0 0
G G ft ft G
0 G 0 ft
0 CO II 0 G (0 0 0
CD 0) 0
ft £ CQ 0 G
Eh X £ o
0 o 0 0
> 1 0 . CQ 1—1 0 • co '— ft G
s co 0 f 0 1 II ft
w 0 CO ,—. 0 • 0 •H 0 GC
w G 0 <
co 0 0 0
+ 1 G < £ CO G — 0
G ■H 0
ro ft b 1-1
CD 0 c ft
£ G 0 i—i
0 0 ■H i—1 O
CD cn ft ft 0 o
G 0 0 0 ft •
fD G £ G o o
0 ft ft V
w > G
CD 0 b 0 II ft G 0 0
1—1 0 0 c ,—. X
rd ft ft 0 Eh X
> ft CO 0 X
49
ro CO CM *o * CD 4c -cf 4: CO i—i rH 1—1 — H 4=0 * O 4c O *
ft « • • • • • • 0 H +1 + 1 + ! +l 4-1 4-1 4-1 13
U vO ro o •H \ CM ft r- LD CO H H 0 G
pH II XI 0 u CM i—i rH r—\ H r—1 i—1 ran i-1
u il 2G 4G g 4-- 4= 4c O 0 U 0 -* * 4c 4c > i—1
•H [> r- cD CM CO CM 4c O rd •' a 4-1
-p • 1-1 '—i 1-1 f-1 o H 1-1 2 6 0 0 (D G • • • • • • • 0) D P G ■H + 1 + 1 + 1 + ! 4-1 4-1 + ! O1 •H >i G 0 i—\ E CM •d1 LD o ro O ID rd •n XI G 0 X u \ ■d1 !-1 CO cD -M1 ro ro G • G 0 rd H
0) u !-1 0 0 0 co G -p
e CM CM 1-1 r—i H f—1 i—i > *H •H •H 0 >1 rd 0 -p 4-1 G G g • 0 rd 0 P 0 a) • G - = 4c 4c 0 G G X C CD *H O O CD i—i CO P -p G II 0
-H 13 > 6 CM CM CM H i—1 r—{ H -P ■H G 0 > G ■H i—i \ + i + 1 + 1 +1 -H + 1 + 1 X) 0 •H i—1 l-1 P G u a O 00 CO ro ft ST 0] U ■P u 0
0 D CD LD 1—\ oo cD CM CM -P G G 0 D G 1—\ CM CM f—s H !-i ‘—! 1-1 G 0 0 G 0
G ft 0 -P O 0 *H 0 u m rd X G 4-1
■H o CM o ft CM r- 0 E 0 0 O 4-> 13 • « • • • o G P *H 0 0 G r—! rH rH o O f-1 • ft >h •H i—1 -P G g a) ft +1 + ! + 1 • 4-1 + 1 4*1 0 xs X! 0 0 4-1 -p \ LD cD s—-i !-1 ft co G rd 0 G G c 6 i—1 tf » • • + ! • • • 0 CO 0 -P II 0) P O CD co i 1-1 i—1 f—1 O ft ft 13 -P ■H G u ■H ft 6 o O o o o ft ft 0 0 rd 4-> 0 2 1-1 G 13 rH 1-\ rH f—1 !-1 •H 6 U O H o 0 0 G D1 to 0 G O o u w 0 G rd G 0 \ »
a •H i—1 *4-1 0 !-1 o 0 44 4c 4c 4c G a U V
e 0 CM o ro o r~ cD i—! P II 0 u a 0 ft rH 1—1 H H o O o 0 X) ii Xl 0 c H G 2H 4c
i—i 0 u + i + 1 +1 +! 4-1 + 1 4-1 -P X3 rd MtG 0 4c ft -H \ a? "vF ft co ro cO ft- o G 0 2 U 0 0 4c
ft co LD LD -oH CM O O ft 0 G a \ 1-i G G d) ft O •H 0C 0 0 u u i—! f-1 H r—1 H 1-1 o rd • 20 G CM
0 0 -P o U 0 O G X • ,C 12 0 0 O 0 0) G 4< - 4c Eh O 0 •' E l-1 •
•H ■H CO CM LD CM CO CD ft 6 E 0 0 o m rH E 1—I 1-1 1—\ i—i O 1-\ O CO •H P 0 V P rd \ . • • • • • • • -p -p *H f—1 0 a
4-) G • +1 + 1 4-1 4-1 4-1 4-1 4-| • G 13 a 13 G 0 rd i—\ LD CD CM r—1 co ft ro 2 0 -p 0 -H 4c
■H •H ft 6 CD r- ID ro 3-1 H ft E rd w ii G -p u • * • • • • • W 0 i—1 0
PQ o i-1 !-1 1-1 i—1 i-\ i—1 o • G XI 04 CJ r—1 ft <0 w P 0 o a a CM O G • CO 4-> 0 o
1 44 G - - 4c 4-1 rd G G •' • ca *H 00 00 CM CO i—! CM H Cl) rd O 0 ii o
ft > 6 rH rH CM !-1 i—! CM i—1 G 6 -P •H u V 0 G rd \ + l +1 + 1 + 1 + ! + 1 4-! rd co -P G H a ft (0 ft y s—i CO CD H O ■vT 0 0 0 0 u
D 0 CM CO o l> LD cD CM E G co G G u 4-1 CD 3. CM CM CM H r—\ H H ■H 0 U 0 O O 0 r—1 £ X 0
C O ft O CM CM r—1 G 0 (1) H G -P f0 • • • • • • • rd CO CQ o 0 LD o G cH o H H !—! i—1 i—\ rd a rH ■H O CD rd • ft +1 + ! + 1 + 1 + ! 4*1 4-1 W X2 o G 6 -p .
4-1 0) rd \ CO cO ro CD cD P0 0 i 0 P 0 O 4-1 1-1 ft y • • • • • • « P ro ca P *H G V ft o ft 0) H ■H 13 0 a
E ro ro co co ro ro ro rd 4-1 rd G 0 X r—! (—s H i—1 H H i—! > 0 2 P 0 0 -
w rH CD o o o o
w ft G o o CM LD CO ft (D P ft o CD ft 1-1 H H CQ E G C co 1 1 ! i i < •H H 0 1 O o O o o E-i Eh 2 u o 00 CD ft CM LD
1-1 e—!
50
4c
4c G • 4c 4c 4c 4c 0 £ o ft) 00 ■vT O G G
f—1 1—1 1—1 i—1 1-1 1-1 l-1 0 G 0 + 1 + ! + 1 + 1 + 1 + 1 + 1 rG £ G 0 ft CO ft CO i> o o P P
0 H >1 • *H
- P u S'? ro i—\ ft i—i o ft ft 0 03 o 0 • G 0 \ CM CM C\1 CM CM i—1 1—1 P 0 0 I-1 0 ft ft G 0 0 0 1-1 o
*H u 0 -P £ -P 0 o 4-> ft 0 0 •H 0 G • ft) 0 0 -p ft 0 o G G ft •H V
ft G ft c -p t>i -p G 0 0 *H 0 G u ft CL) -H G G 0 0 U ft P 03 G G 4c
G 0 13 03 0 *H ft 4c
0 G 0 -P G 4c
U U *H 0) G P II X ft ft ft 00 CO ft co G 0 0 ft
ft) 0) • « • • • • • • 0 G -P II H £ G CM CM CM !-1 1-1 rH i—1 ft •H 0 o CM CO ft ft •H + 1 + | + | + 1 + 1 + 1 ~H 0 > \ O rd ft) u £ CO co !-1 ro ft) in 1-1 -P 0 ft ft o ft a \ • • • t • • e 0 03 0 D u •
ft o l—^ ft ft) ro !-^ rH o G 0 £ o ■H £ CM CM CM CM CM CM CM -P • - •s
C ft C 0 G G 0 V
o a N 0 -P 0 0 0 tt) a G ■H •H G ft
c g 0 0 -p 0 0 ft 0 £ p 0 G 4c
•H rG 0 ft G 0 4c
to 13 Eh G G G 0 P G P •H G rH ft a) 0 0 u a • 0 03 a ft 0) CO ft • 0 X G £
a G • • « ft O oo i—! s £ O 0 P • 1-1 CQ c > f—1 i—1 CM • • • • • 1 a •H £ O ft a) ft £ 1—1 i—I 1-\ ft 00 co CO w 0 ca 0 P • O G D \ + 1 "f 1 + 1 + | + 1 + 1 4-1 • G £ 0 -H o
1 rd ft ft) CO O rH ft ft ro U1 •H • G 0 0 V ca a) 0 o ft o ft o r- r-
+\ i—i 0 -H P 0
f~“! i—! rH 0 ft 0 0 0 ft no a 0 0 ft P ft G 0 • 0 0 4c
0 03 0 -P II ft ft G 0 *H 0 ft 0 0 - £ ro 0 ft o p
-H cd r~i r—i 00 oo CO co 0 0 -p ft o t—1 rH o o o o 0 ft G 0 .V • v
a a) ft • • • • • • • G 0 P £ G c in (D g ft \ 4-1 + 1 + 1 + 1 +1 4-1 + 1 0 •H 0 0 0 o ft u ft ft CO CO ft CO r—1 ft) oo 0 03 0 •H •H • ft X 0 1—1 O ft ft ft) LT) 0 ft 1-1 p p o W 0 £ • • • • • • • 0 0 G ft 0 0 V
'ftp ro ro co co ro P G 0 G G H 0 -P II a U ft
• 0 > 0 ft X X LD w r—H o o > 0 £ ft 0 0 -
0 0 o LT) CO W ft g o o CM r—1 i—j ft 0 P ft o ft) ft rH l i ft £ G G CO 1 1 i o o <c ■H -H O [ o o o CM uo &H E-* s u o ro ft) ft 1-i i—!
TA
BL
E
6.
Effect
of
Na
g-O
HB
in
fu
sio
n
on
ven
ou
s
pH
, calculated
an
d
measured
bicarb
on
ate
co
ncentration,
an
d
ven
ou
s
carbon
dio
xid
e p
artial pressure.
51
o g p 0 0 (D G ft
g 0 P P 0
0) rd ft £ p P G
>1 0 Cfl P ft
cO i—1 f" CM Ud o p rd CM ft • • • • • » • C (D 0
o X 1-1 CO CM CM CM CM CM 0) P P u \ 4* 1 + 1 + 1 + | + 1 + 1 + 1 CQ 0 •H
a £ CM i—1 1-1 Ch cn Od CO 0 X -
£ p "vT co ro ro G ft • 0 > ft G o •H
CD •H p G G 0
P £ G P P •H 0 0 p P
•H P G g 0 p 0
0) CD G 0 O -P - £ = ft •H 0 o cn CM O i—1 CM CM rd P 0
P C • • • • • • • i-1 P 0 P 0 0 ft i—1 o 1-1 1-1 i—1 1-1 1-1 0 P P 0 G P \ + 1 + | + 1 + 1 +i + | 4" | g 0 G G P g ft CO CM r-~ o ro 00 p 0 0 •
0 0 (D • • • • • • • g 0 P > 1-1 0 O £ Dd 1-1 CM CM CO 0 P co o CD -H 1—1 CM CM CM CM CM CM 0 G II t 2 P 0 o o
CD £ 0 CM ft 0 £ O V
Eh G o ft P c a 0 0 X
(D • rd ■H > P P • 0 0 0) 0 - 2 £ P •V
P C o O UO CM CO 1-1 1-1 • P ft • V
0 0 • • • • • • • w 0 c ft CM '-1 rQ 1—1 1-1 1-1 1-1 1—1 1-1 1-1 • G •H O P G ft + 1 + 1 + ! + 1 +1 + 1 + 1 cn •H p * O 0 \ CM Ch lid 1—1 ■vf CO i—1 m 0 o
i—1 o ft +1 0 ft 0 V rd -H (D o o i—1 1-1 CM CM CO co o 1—1 ft U P £ CM CM CM CM CM CM CM c rd i p
rd P CQ. Z
0 0 £ ro 0 p
ft o - 4< X z CD 44 G
CM CO CM CD ■X CO G 0 • 0 • - '-1 O 1-1 1-1 O 1-1 o rd 0 > Ud o O O O O o o 0 •H o
• • • • • • • 0 ft 0 II •
ft +1 + 1 + 1 + 1 + 1 +1 +1 CD rd 0 o ft 1—1 CO ro co o CO CD P G G ft V
ro CO ro CO CO CO CO i—1 0 P ft ft > • e • • • • • rd > ■H
t"~ o o o o > rd p > -
0 i—1 o o 0 0 O Ud 00 P G o O CM 1—1 *—i
0 P P O CD CTi i—1 1 i £ c G co 1 1 1 o o
■H •H 0 1 O O O CM Ud 2 CD O co CD Od i—1 i—i
TA
BL
E
7.
Effect
of
Na
B-O
HB
infusion
on
pla
sm
a
levels
of
glu
co
se,
in
su
lin
,
glu
cagon
an
d free fatty
acids
52
d •H o G id O
•H >1 a o o LD o 0
4-> cd co CM CO a: "0 a G 2 +1 + 1 + 1 +1 4-1 rd • rd 4-) G o o O o 03 o
rd a G r—1 OH r- o C/3 4-1 £ 0) CO CM CM CO 4-1 C/3 03 03 0) 0 C £ (0 0) G CD a •H
i—1 G O C/3 G a d M-l O 03 •H
G G a a G rd CD d G d
d 03 G d 03 a O 0 C G
•H •H rd a G rd a rd 03 4-1 C/3
G G l—1 at! O a £ 0 C/3
rd O' G \ 'vO CO LO 1—1 rd £ rd CD O' + 1 +1 +1 +1 0 C/3 > 03 o O a LO cm CO o G 4-1 CD G C LD in U0 LD 4J G C
1—1 rH O G 03 0 a Oi O 0 £ ■rH
O 03 C/3
G G G 03 G a O d C/3 c
•H rd •H a 03 id cO o LO LO CM £ CQ G rH • • • . • • • • X
G 4-) £ 1—1 o i—i 1—1 i—1 1—1 i—1 s 03 O •H G \ +1 +1 + 1 + 1 + 1 +1 + 1 H G 1
£ i—1 0) D o r—1 CO CO 00 CO *H oa 03 G O CM CM 1—1 1—1 i—1 1—1 i—1 i—1 rd rd 03 G + 1 03 a
i—1 G O C/3
a -H O G cd •
rd d C/3
03 •H
G £ co 03
O 03 -H 03 a G 4-1 G 0 G rd cO CO CM G> CO 00 r—1 rd -H
G • • • • • • • 03 d 0) 4-1 rH CM CM 1—1 1—1 o i—1 C/3 a
rd 03 G Oi + 1 + 1 +1 +1 +1 +1 +1 03 rd G £ O CD £ Ch r-~ CO CO l> LO CO G G 03 03 0 O 00 CO CO 00 CO 00 00 1-1 03 a II) G G rd > rd
t—1 i—1 O > rd £ a Oi O
03 1—1 o o 03 0 o uo CO a G o o CM 1—I ‘—1
03 G a o CD OH 1—1 1 1 £ G G co 1 1 1 o o
•H ■rH 0 1 o o o CM L/0
EH 2 o o CO CO OH 1—1 1—1
£ 00 m CO _ o
-H 1—1 CO i—i CO (N ro CN
£ + 1 +1 +1 +i + 1 + | +i + 1 r- 00 CO o i—1 r" 40
o uo o CO 1—1 LO 40 CN
LD 1—l LO i—i CN
S l 3- O
£ o
*H -p
rtf £ ft £ £
•H £ LO
i—1 r- 40
<—1 40
1-1 CM ro
o CN
o + 1 + 1 +1 +1 + 1 +1 + 1 + 1 cr\ oo 00 00 0> CN m cD LO
l in '—1 00 1—1 LO 40 CM CN
30
i—1 m '—1 CN
a
rtf
o
a ■H
£ rtf
rtf £ £ w £ in
CM
i—1 co
O
CN
1—1
CM CO
CO
CN 0 + i + 1 +1 + 1 + 1 +1 +1 +1
1-1 o N1 r—1 o in i—1 vD o o ft co 40 CM CS0 i—i LD O CO CM
on
1
o i—1 in i—1 CN
£
O
£ ft £
•H
i—I
m o X £ LO o CD 00 o -P co i—1 CO 00 i—1 CM CN
1 £ + 1 + 1 +1 + 1 + 1 ■H + 1 + 1 aa 0 CO CM cr> o LO Ch in
U in i—1 00 CM N1 CO CM CO 0 i—1 in 1—1 CM z
ft 0
ft 0
0) 4-1 ft 4-1 ft ft O
ft 0 0 ■H 0 0 £
• U £ a ft H 00 0 0 •H •H 0 0 i—1 0
£ £ 0 £ £ £ i—1 £ w 0 0 £ 0 0 •H 3 •H ft £ £ 0 ft ft i—1 U O ft •H £ £ £ £ £ 0 -P <C £ 0 ft 0 i—I l-1 £ *rH r—1 Eh < Eh ft CO O 0 ft CJ u
53
o in CO
CO CN 40 LO m 40 40
o 1—1
+1 + 1 + 1 + 1 + 1 + 1 +1 + 1 + 1 CO CM i—1 co CO CO r- LO co CN CO r- CM N1 co CM CM CN i—1
£ (D ft
0 rtf ft £ -P
K*1 0 ft ft 0 £ 0 0 ft w W 0
r-H CM o £ ft> • CO CM 40 ■N1 LO 40 in 1—1 ft £ O
+1 +1 + 1 +1 + 1 + 1 +1 + 1 +1 0 -H 40 1—1 CO ft* 40 CO in o ro £ £ 0 O CM CM t'' CM in N’ "N1 CM £ £ CO CM 1—1 43 ft ft
0 ft •H ft £ 0 ft 0 £ 0 ft ■H
0 ft I-1 ft 0 0 ft ft £ 0 £ ft 0 £ 0 ft
CN CM 1—1 O ft 0 co CO 40 40 40 40 1—1 0 £
+ 1 +1 + 1 + 1 + 1 + 1 +1 + 1 +1 0 0 CTi CN in o CO CN in 40 o 0 £ 0 i—1 CM CO o CM in -M1 N* CM ft 0 £ CO CM 1—1 ft £
£ £ co O
• rd *h • (D w
S £ £ • 4-1
H 0 £ • £ ft
C/] -H i—i m
+ 1 0 z cn O
£ rtf |
CM ft i—i 0 ft CQ. CM CM CO CO 40 40 40 i—I 0
+ 1 + 1 + ! + 1 +1 +1 +1 + ! + 1 £ CO 0 cn in CO ■N1 ■nT CO in 40 40 z in CM CO [> CN in i—t 0 ft CO CM i—1 £ 0 •
0 0
0 ■f—1 0 to 0
0 0 0
£ £ £ l-1 0 £ rd > ■H > 0 ft
0
ft 0 £ •H £ a •H 0 0 £ 0 0 £ £ 0
-p ■H ■H 0 i—i 0 a 0 £ a £ 0 0
£ •rH 0 >H 0 £ -H i—I £ •H £ £ u •H 0 0 •H
£ •H ft i—i 0 £ a 0 ft i—1 CM ft 0 £ 0 £
i—i £ 0 \ 0 0 >h ft 0
< CQ > 1-1 s H Eh ft
TA
BL
E
9.
Effect
of
so
diu
m b
icarb
on
ate
infusion
on
urine
flo
w,
creatinine
clearan
ce
urine
pH
, and
am
mo
nia
an
d titratab
le
acid ex
cretiin
54
> < Pi
G •H
s \ a 0) a
C > -H
co S
X X
u
G o
£ o
I—I
44
CD G
■H G P
\ a CD a
X a
c •H 6 \
i—I
£
CN CN i—1 o i—1 a o
in ■a1 n n n CO a
+ 1 + 1 + 1 + 1 + 1 + 1 +1 in o n i—l cn o t>
• • • • • • • co i—1 '—1 CN o CN r—1 0 co co co co CO ro co 1-1
b 0 b
13 0 0 • %. G G 0 b
o o cn r-~ vO cO n ■H u ■H 0 0 G b
CO r- cO n CO n b b 0
+ 1 + 1 + 1 + 1 + 1 + 1 + | G b G II oo ro r- ro a1 n 0 0 0 <
• • • • • • • 0 0 E CN CN cn n a co ■a1 0 0 1-1 P < cn co co 00 CO r- g b 0
a G 0 0 0 G G h G 0
0 ■H •H 0 G c b P P *H 0
1-1 m b G 0 0 0 0
r * > 0 0 X cO o 00 cn o 00 co 6 G 0 O i—1 o o i—1 o o 1-1 0
• • • • • • • 0 0 0 + 1 + | + 1 + 1 + 1 +1 +1 g G II *H a CO CN cO rH o o b •H G co o CO co cn o 1—1 G i—1 G 0
t • • • • • • 0 0 U P n n n n n cO CO 0 0 U fc
0 b b
0 b
(D >
•H 0
0 ■H cn 0 G
Li G 0 G
i—l
s 0 P G o r- CN M0 LO O • 0 ■H P •
+ 1 + 1 + 1 + 1 + 1 4-1 4-1 W 0 13 II o i—1 o CM co co ro ro • 0 > V
■a1 ro ro CM LO LO P G co a '—1 i—i •—1 1—1 •—1 •—1 i—1 0 0 X *
+i b X CO 0 P
G £ • 0 b G 0 0 Li X 0 6 13 a •H
0 0 b 0 a 0 0 0
n 00 G 0 b c G • • • ro 00 kO M0 0 G 0 ■H 0 CN
i—\ CN rH • • • • 0 G X O +i + 1 + 1 i—l 1—1 i—1 i—1 0 > a P 0 .
■a CM + | + 1 +i 4-1 0 0 G o • • • 00 ro i—1 MO P •H II 13 V
i—i i—i O • • • • 1-1 0 G X •H a •—i i—i i—1 cr\ o CM •—i rO b P a 0
1—1 i—1 i—i > b 13 p 0
0 rH o o 0 0 o n co b G O o CN i—i t—i P b o co cn i—i l i G G co 1 i l o o
•H 0 1 o o o CN n s u O co cO cn i—1 i—i
i—1 co in r- cO 04 * • • • • •
G r-l
oj
+ 1 o
nj
+ 1 o
CM +1
CM + 1-
CM +1
CN +1
CM + 1
£ r> G1 CTi o
\
27 .
i—1 ai r~ cO
2 o
i—1 £- 2
4
CN CM CM CN CM G 0
£ P
o ,—| i—1 1—1 oo in •H ■H
CM
+ 1
,—|
CM CM 1—1 1—1 i—i 4M 0 £ 0 2l • H
i—i
+! + 1 + 1 +1 +1 +1 0 P P 0
0 £ \ 01 0
LO CO G1 LO CO 10 ■H G 0 0 CM 1—1 1—1 o 0 G 03 0 G g o
i—1 i—1 1—1 1—1 1—l 01 -H 0 -H 0 • •H i—i 0 0 G a 0 0 ,—| CO co G 0 m 0 0 P
> a
3- co i—i i—1 f—1 o 1—1 0 o II 0 0 G O • CM • • • > u > G £ 0
' + 1
. 40+
.
+i + 1 +1 + 1 +1 0 X 0G 0 0 G iG o O o LO uo 20 0 0 0 \ ■<+ CO 1—1 1—1 CTC 0 0 G O 1—1 0
•H £ a
O1 lo . . • • . rC 2 G a1—i G P 0 •
'st1 ■n}1 CO -P >. 0 0 0 •H a £ . G 0 n 0 in
r- ,_i CM 1—1 o 1—1 o 0 0 0 2£
G w 1-1 1—1 1—1 1—1 1—1 -P -H •H 0 a p G 0 G i—i • i • • • • • G 0 G 03 ■0 0 -p •H
+ 1 +l o
1
+ 1 +1 +1 +1 +1 0 0 0 •H 0 U 0 £ CO O'* l> o ■01 0 0 0 0 0 0 G a \ G1 00 o co co CO 0 P G 0 0 0 0
03 1-1 i—i i—i 1 1 • . . . , 0 ■H G i—1 G G
0 u £ 04 CM 1—i 1—1 1—1 1—1 i—i 0 rC 0 0
0 c o 0 0 0 0 a 0 0 0 G 0
£ G r~
oo cO 1—1 CO o 0 0 0 0 ii 0 - •H
+1 LO
CO CO CM CM 0 -P 0 £ i—i 2 0 0
> £ co + 1 + 1 +1 +1 + 1 p 0 0 0 O
1-1 03 \ + 1 i—1 o CO co ■*P i—1 £ 0 ft -H 1—1 tr CM CT> 00 ■01 uo ^P rd P 1-1 0
0 u 0 04 1 1 CN •—1 1—1 1—1 1—1 i—1 > 2 •H a 03 g
G 0 D n CM 03 03 •H 0 0 1—1 •—i 0 0 II M -H
G G CN •xP 0 0 0 i—10 G G 0 CM . ■ • • • 0 G OH 0 0 •
CM o o o o o -p 0 0 a rG 0 ■H ' O
+ 1 +1 + 1 + 1 +1 + 1 c £ 0 0
w £ /'“'I LO LD LO o in 0 0i 0 • ^ X p p
G o V—J
+1 CM
• 1 • • • 0 G 0 0 II 0 m -H • i—1 o o 00 •H 1-1 0 G 03 i—i \ co o o o cn oc 0 0 a g >h£
•H 0 o o1 o \—J t-1 1—1 1—1 1—1 rC P 0 UG
0 0 a 0 i—i Eh 03 ii 0 U G
4-1 £ 00 0
G CM
CN LO 1—1 o o 03 20 • - 0 0 O ,—j i—1 1—1 1—1 i—i i—i • 0 a 1-1 C 0 G
0 i—1 • • • • . • G 0 0 G
0 • o o o o o 2 •H •0 0 G 0 G o
+ 1
<—* + 1 ^p
+1 + 1 + 1 +1 +1 0 • £ g a 0 c
0 •H CO cO co 1—1 1—1 w -P o P 0
0 0 £ 00 co o CO <T> G •H 0 II 0 0 2 \ LT) • . . • CO 0 0 03 0
■H 0 O i—! i—1 i—1 i—1 1—i o O o £ 0 x >
G 0 £ + 1 0 •H 0 0 a f—1 0 G D
£ 0 G P II 0 P
03 G
M0 LO 00 CM i—1 rH 0 i—1 G 00 •-
■H >
•0 ,—| i—1 1—1 r—1 i—1 rH 0 rd 0 2H C 03 G £ i—1
+ 1 + 1 +1 +1 +1 + 1 £ > O 0 o 0 0 0 \ + 1 o CO •CP o Gc 03 O -0 in 2 01 O (J\ O' r—1 CM CM 0 0 0 • v i—1 G
ip ' D 0
3- r0 ,—| 1—1 1—1 1—1 1-1 1-1 0 G G G g: 0
G CM 0 ■H 0 0 0 0 0 0 •CP CN r- rH 0 •H G
■0 CM • • • • • 0 0 G G II G -p -P • o o o i—1 o 0 0 0 0 0 o 0
G i—1
+1 + | LO
+1 + 1 +1 +1 +i p 0 0 > 0 0 P
0 o LO m in o 1—1 G 0 0 1-C
4-1 0 \ CM . 1 • rd 0 X 0O 4-1 W
X
0 2
a tr 0 CO CM
rO
CO
CO
co co
CM CO
CN cn
•01 CO
> CO S 0 2 0
£ co G i—1 1—1 1—1 1—1 i—i 1—1
O o o p—1 0 1—1 o LO CO
w 0 0 o o CM i—1 1—1 -p 0 CO
| CTC
1 1—1
1 | 1
PI 0 P -P O o o
CQ £ G G CQ
I o o o CN LO <C Eh
•H Eh
•H £
0 0
1 O CO co ac i—1 1—1
55
<0.0
5;
" 0
<0
.02
; *
p
<0.0
1.
TA
BL
E
11
. E
ffect
of
sodiu
m bic
arbonate
infusio
n
on
ven
ou
s
pH
, calc
ula
ted
and
measu
red
pla
sm
a
bic
arbonate
concentr
ati
on
and
ven
ou
s
carb
on
dio
xid
e partial
pressu
re
56
oc f\l • • • •
O CM ro CM o o o o O + 1 +1 + 1 +1 4* 1 + 1 “1" 1 a O' CM CM O' ro CM CM > CO 'M1 ro *CF
(D P rd c P 0 c 0 p 0 -X C p •H = z •X -X •rH id p co V1 ro CO uo CM CM 0 rd o rd 1-1 p P
P •H P K. o o o o o O o c P 0 p P D1 +1 + 1 +1 +1 + | + 1 +1 0 0 P G 0 in CM 1—1 CO cO o 0 0 rd 0 g • • • • • • • 0 0 0 6 u <30 i—1 ro P1 LO cO p P rd 0 G 1-1 CM CM CM CM CM CM a G 0 rd 0 0 0 s i—1 0 p g
a 0 0 P G G
o i—1 0 p rd rd rd G > 0 0 O b 0 ■H i—1 P p 1-1 cO CM CO cO in cO 0 0 •
P p rd • • • • • • • p G CM <D rd P J 1-1 1-1 1-1 CM 1-1 o o p •H O P 0 P \ + 1 + 1 + 1 + 1 + 1 + 1 +1 G i—1 O rd •H C a in in Md -cF ■cr o 0 0 •
1—1 p 0 0 • • • • • • • 0 0 o 0 O 6 Ch i—1 i—1 ro CM cO ^f rd V 0 rd G i—1 CM CM CM CM CM CM 0 P • a
1—1 g 0 P 0 rd 0 0 P 0 -X O rd > 0 -X
i—1 « N •rH 0 a • 0 p
s 0 G • 0 -H
W 0 p • • • 0 1—1
-X -X V) G p o CT> Z CO 'cr -X 0 0 •
o O o CM rH o lO + 1 p o o O o 1-1 i—1 o 1—1 co rd V
• • • O • • • c £ a o o o • o o o rd p •X
a +1 +1 + 1 + 1 + 1 +1 + 1 0 O a o •^r ro cO O' 00 g P
co CO CO ro ro CO ro 0 rd > • • • . • • . 0 a 0
o- o- O' O' O' O' O' P rd P • V
rd P 0 CM 0 o
0 > a • 0 rd G o G •H V
1-1 0 p rd ,C G a > P P
0 i—1 o o 0 0 o UO CO p p o o CM 1—1 <—t
0 G p O CO co 1—l 1 1 g C G co 1 1 1 o o H •H 0 1 o o o CM UO a 2 0 O co cO o> 1—1 1—1
TA
BL
E
12.
Effect
of
NaH
CO
^
infu
sio
n
on
pla
sm
a
glu
co
se,
insu
lin
, g
lucag
on
, free
fatty
acid
and
keto
ne
bo
dy
concentr
ati
on.
57
CQ EC O
i i g d CQ g 0 o o LO o £ "t~ a) ■H co co CO co
to O a 4-1 2 + 1 +1 + 1 +1 d < g d 41 CO o cn CO
i—l o 0 g 04 o i—i cn ft < o 4-1 1—1 i—i T)
0 g
-H
i g d ro g 0 o o -P £ tu •H i—i cn ft CM w n co u u 4J s + 1 +1 +1 +1 -P 0 d < g d CO 1—1 CM LD g
i—i o 0 g CO CO W co 0) to ft c u 4-1 w -P
(D g g 0
i g ft £ rd g 0 O W1 O CO <D 0 £ CQ <u CN CO CO 1—1 g g id EC u 4-1 S +1 + 1 + 1 +1 p rd O g d 41 CO cn r- CN 0) to
i—i 1 0 g cD CD CD P d ft CQ O 4-1 i—1 0
(0 £ >
0 i g UO W) CO LD i—! g
rd g 0 CO Nf CO 0 •H
£ (D •H + 1 + ! + 1 +1 g '—1 to o 4-1 2 o O o U0 44 0 rd < g d g o i—1 cn O g to
i—1 ft 0 g CO CO CM CO 0 d ft (A u 4-1 o JQ
g 0) 0 0 i g & >
rd gi g 0 i—i Eh ■H £ d CD *H £ CO CO W1 CD CO to o u 4-> \ + 1 + 1 +1 + 1 to rd p g d Oi o cD 1—1 O • 0
1-1 i—1 0 g ft LD W1 UO UO s U ft Cn o 4-1 ft 0
ft p to
+1 g i g CO
rd •H g 0 i—1 CM i—1 1-1 1—1 CN CM 1—1 g £ i—1 CU ■H £ + 1 +1 + 1 + 1 + 1 +1 + 1 d CM to p 0 4-1 CM o CO cn CO 00 e o rd CO g d D CM CM i—1 r-H i—i i—l 1—1 £
i—1 g 0 g 0 & •H o 4-1 0 0i
g d d g
0 CD i g to >
d to g 0 'N1 co LT) cD r- UO 0 d £ 0 0 •H + 1 + 1 +1 +1 +1 + 1 +1 P to U o 4-4 Oi o 00 o co CM co r-H 0 d P g d £ CO r- 00 co CO CO CO rd x:
i—1 ( 1 o g > 44 ft 01 u -P
to i—i O o 0 0 o U0 CO -P g o o CM i—1 1—1
0) P 44 o CD cn i—! 1 1 £ g g CO 1 i 1 o o
•H *H 0 i o o O CN U0 EH o o CO cD cn i—1 r—1
o
CD £
•H -p
ste
ad
y
wate
r d
iu
resis.
NaH
CC
U
infusio
n
was started at
c •H 6
i—1 Ch ro o> o ro i—i f—i 1—1 ro 04 LD + | +i + 1 + 1 +i +1 + 1 i—I o> o r-> co cso O
1 C\! 00 CM 140 40 O i—! •—i CM ro
— o> 2
£ O
-P G 0 -H u e
4-4 44 1-1 40 CO
C O 40 i—1 1-1 CM CM ro 00
0 OO + 1 + 1 + 1 +1 + 1 +1 +l 0 1 ■vf 1-1 1-1 CO o> CO 40 G o 140 1-1 00 C\l M1 C"- 0 ro 1-1 1—1 CM ro 0
13 •r|
o 0
0 G
•H
to o 01 1—1 04 i—1
ro CM ro 2
6
i—1
E ro + 1 +1 + 1 + 1 + 1 + 1 + 1 0 1 LD O 1-1 O 00 CO to O 140 •—i 00 CO ro L40 r-'
i—i a
•—i 1—1 CM ro
C O
G O
-H 0 G
4-1 r-| c 0 LD ro ro SP
•H G ro 1—1 40 CM L40 CM ro -P + 1 +1 + 1 + 1 + 1 + 1 +1
ro G O o r-~ 00 ro 140 o O 0 L40 i—1 CO [> ro 40 CO U U i—1 1—t CM ro a: 0 2
o
-p
u 0
4-1 44 w
13 0 • •H 0 G
ro u C ■H i—1 to 0 •H 0 (—t 0 0
G G 0 c i—1 G G W 0 •H 0 C •H G •H *H
c G 0 •H i—1 G O G CQ G G G 0 -P J>i tO < £ to rC 0 G •H i—1 l-1 Eh <c Eh Eh CO O, u o <
58
ro CM •M1 i—1 CO r- i—i CO
+ 1 + 1 + 1 + 1 +1 +1 + 1 +1 u0 o r—1 i—1 O CM L40 ro CO LO L40 OJ CM
CM 1—1
13 0
4-1 G 0
0 4-) rC 0 ■P l3
0 0 O 0 G 0 CM CM L40 r- i—1 CO -p •G £
+1 + 1 + | +i + 1 + 1 + 1 +1 c 0 CM o> i—1 ro 04 40 i—1 oo 0 -P G ro CM 00 140 ro 140 rO CM w 0
CM i—1 0 0 •H G 0 a 0 G 0 -P 4-1 G c G
0 H
0 6 G 0 ro
i—1 G O tO G U > 0 2
0 0 i—l 0 2
ro O 0 E i—l ro (J\ ^P ro 40 •—1 140 G 0 + 1 + 1 +1 + 1 + 1 + 1 +1 + | -P 0 43 i—i CO 140 140 ro 1-1 i> 1-1 G c -P ro ro O 140 ro LD CM CNJ 0 •H
CM i—1 0 i—1 •
0 0 0 0 •H
rG 0 0 Pi 43 0
G 0 G
• > *H 2 13 W 0 cn 0 G
0 0 CM + 1 0 -P
CM ro r- f" 'vT i—i LO 0 0
+1 + | + 1 + 1 + 1 +1 ~f~ 1 + 1 G G £ 00 CTi 1—1 i—1 'sp r- CN to 0 CM CM LD ro CN 1—1 0 rG
CM i—1 6 ro 13
0 44 0
13 0 G •H G •H
0 •H 0 G 0 0 0 C 0 C
-p •H 0 (—1 H 0 0 0 G 0 0 G
■H 0 G ■H i—1 G 0 G G U 0 0 rG ■H •H rG •H l-1 0 c 0 43 40 i—1 CM 0 G 0 G 4-1 G rtf \ 0 rG 40 0 0
03 > 1-1 H Eh a 2 2
TA
BL
E
14.
Effect
of
Na.
Lacta
te
infusio
n
on
pla
sm
a lactate
an
d
keto
ne
59
Q) £ 0
4-J 0
ft! L rd
0 u o 00
e 4-> 2 <o 1—1 1—1
co £ ZL + 1 +1 + 1 +1 0 0 r^- 04 ro ■cT
1—1 o i> ft 00 ft ft a mi
0 0 mi rH u £ ■H
0 CO •H 0
4-J 4-J fd fd
0 £ 4J
&H 0 ft u
CO 0 ■H
(D U c u co Li
0 ft 4-J 4-1
u *H 0 £ P <c 4-J u <D ft o fd e >1
< u 0 QJ X 4-J 0 U 0
0 a 2 ro CM ro CO 1—1 P u e 0 ZL +1 + 1 +i + 1 ro CO m)
co o ft CO ft CM > fd Li
0 £ 1—1 i—1 i—i CM QJ ft l—1 0 1—1 e 1 ft u 0 ft
u QJ II 4-1 a ft •
c £ •H ft CM
m 0 0 i—1 O o X ■H o QJ 1 o o 4-> CO ca •
1 rd 0 rd o ca L ft • V
4-) 2 i—1 LD EH co ft fd a CO CM ft i—1 0 ■H e 0 +1 + 1 +i + 1 > CO -X
co O CO ft ft 00 • •H QJ •X
rd £ LD LD cO ft • CO H '—1 0 2 CO P ft o • QJ ■H •
ft 0 ft ft • a •H 1—1
CO p U a o co QJ fd •
o +1 ft o 4-J £ co fd a V
rd 0 £ £ •H ft 4J 'H -X -X rd ft ft O ft -X -X - -X •X - 0) 0 Li 0 -X
• fd fd LD o ro LD LD ft e ft a £ i—1 L • • • • • • • QJ rd fd 0 4-J o i—! o i—i O O o o ft QJ 0
-H <U a 2 + 1 +1 + 1 +1 + 1 + 1 +1 u fd ft ft • -
-U e o 6 ft ft LD ro o o 00 fd u CO 0 LD
0 co 0 • • • • • • • aj u O
L rd c cO ro CM O o o ft CO > ft 0 •
4-> rH 0 1—1 ft i—1 rH 1—1 0 rd £ II o £ ft o P •H 0 V
0) i—1 QJ U < ft O rO ft P 0 c > 44 ft < -
CO i—1 o o 0 0 o LD CO ft 0 o o CM i—1 r—1
0 p ft O co 04 1—1 1 1
6 £ G co 1 1 1 o o ■H •H O l o o o CM LD
Eh 2 O o co co 04 i—l i—1
TA
BL
E
15.
Effect
of
Na
lactate
infu
sio
n
on
ven
ou
s
pH
, calc
ula
ted
an
d
measure
d
bic
arb
on
ate
concentr
ati
on,
and
ven
ou
s
carb
on
dio
xid
e partial pressure.
60
UO CO O O CO CO ft
CM ft o 1-1 1-1 1-1 1-1 1-1 1-1 O \ + 1 + 1 + 1 + 1 1 + 1 + | u p CM CO CO CO CO G ft £ V 'tr 0
•H
> ft -p G 0
•rH G G ft P G
0 ft 0 ft O
- * * ft ft G ft UO CM CM CM CM co o 0 0 (D — CO G 0 G co ft 1-1 i—1 '—1 1-1 1-1 o 1—1 ft 0 O \ + 1 + 1 + 1 + 1 + 1 4*1 4* 1 c 0 0 0 U ft CO i—1 CO UO CM UO 00 0 ft ft 0 ft CD • • • • • • • CO ft 0 CD 6 CO i—1 CM co UO UO 0 0 C 2 1—1 CM CM CM CM CM CM u 0
ft CO ft 0 ft G G G 0
0 O 0 E P 0 ft
ipH G ft 0 P 0
CD - > 0 e ft CM V* CO CM o 0 w 0 ^ • • • • • o • 1-1 0 0
rH CO ft 1-1 1-1 i—i 1-1 1-1 • 1—1 0 e 1-1 0 O \ + 1 + 1 +1 + l + 1 1—1 +1 g ft O U ft ["■ o 1-1 +1 o ft 0
i—1 ft CD • • • • • • C G 1! o 6 o CM V’ CO V UO 0 *H u CM CM CM CM CM CM CM 0 i—1 —
QJ co v 0 0 O ft POO Eh ft ft -X
CD • >
2 •r| 0 W 0 -H CM 01 0 0 O
+ 1 0 0 • o G o u P V
- - * * * c P -H ft CO CM I> 00 i—1 o 0 0 ft rH i—1 rH i—i o rH 1—1 0 = o o o o o o o E co G
• • • • • • . 0 +1 + 1 4*1 +i + 1 4* I + 1 0 ft ft o CO LO r- 00 00 G 0 0 • ~ CO CO CO CO CO CO CO 0 £ UO
• • • • • • • 0 o r- r- o O r-~ 0 ft >1 •
0 0 ft o P G 0 V
1—5 0 0 ft rd > ft > 0 0 -
H Eh
0 i—! o o 0 0 o U0 CO
ft G o o CM 1—1 rH
p ft o MO CO 1—1 1 i
G G co 1 1 1 o o *rH 0 1 o o o CM UO
2 O o CO CO OC r—1 l—1
TA
BL
E
16
. E
ffect
of
Na lactate
in
fu
sio
n
on
urine
flo
w,
creatinine
clearan
ce,
urine
pH
, am
mo
nia
an
d titratab
le
acid ex
cretio
n.
61
> < Hi
D
> r
£ 2
D
X a
D
0 O
u
£ o
I—I
p
0 d
•H
0 x>
0 g
■H
H
w •H
CN CO CN cr\ o LO 1—1 w d
a . • • • • • • 0 0 -H 1—1 1—1 CN i—i CN CN Q) 0
6 + 1 +1 + 1 +i + 1 + 1 + 1 O' p p
\ CM cO '-1 CN CO 0 •H 0 ft . • • • • • • 0 p 0 0 CO <—1 CN 00 n~ CO LO 0) 0 3. CO CO CO CO CO > 0 X
0 0 0 ft
Q) 0 0 P •H ft d
z z z -X -X >1 0 co oo i> CO o CN P p
d • • • • • • • W 0 g
■H CO CD r- cO LD ft 0 0 6 + 1 +1 +i +l +1 + 1 +1 d ft
\ co LO U0 O' UO O CO CD w II X • 1 • • • • • CO 0 CN CN CO o CN lO CD O' > 3. CO CO NO NO NO LO LO 0 d CO
ft •H X CD 0 X
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18
. E
ffect
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infu
sion
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pla
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cagon,
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free fatty
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63
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64
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65
TABLE 20. Factors known to influence renal ammonia
production and excretion
A. Ammonia excretion
1. Urine pH
2. Urine flow
3. Arterial blood ammonia concentration
4. Rate of ammonia production
B. Ammonia production
1. Substrate delivery: glutamine and other amino
acids.
2. Enzyme activity: glutaminase I, glutamic dehydro¬
genase .
3. Cellular concentration of
a) ammonia
b) glutamate
c) a-ketoglutarate
d) NAD?NADH+
e) potassium
Renal gluconeogenesis 4.
66
FIGURE 1 „ Relationship between gluconeogenesis and ammoniagenesiG in the kidney. After Goodman et al. (119)
t
GLUCOSE
I FRUCTOSE 1', 6 DIPHOSPHAT
U ■
uEPCA= oho sphoenoloyruvate c arboxykinase
67
Alanine
Preformed blood, ammonia
\ -lut amine
/ amide nitrogen
Figure 2. Passive secretion of free base ammonia by non
ionic diffusion. The major fraction is trapped in
acid urine as relatively non diffusible ammonium
(Pitts, 27). ion.
68
Cell
Blood
H
■+
NH3
1 N H +
4
Figure 3 Origin of renal ammonia in
metabolic acidosis.
in dogs chronic
69
figure [|A. Effect of lactate infusion on plasma lactate concen- trat ion.
Values are mean+/- S.E.Li. for 4 acedemic subjects. 3 successive measurements were obtained during steady water diuresis before lactate infusion. These were aveiaged and represented plasma iactate concentration at time 0 (control value).lactate infusion was started at time 0. Each value obtained afterwards was compared to the control value using the paired t-test.
figure IpB• Effect of la b-Ohb infusion on Plasma ketone concen¬ tration.
Values are mean+/- S.E.h. for 9 subjects.la.B-OhB was Started .at time 0. Each value obtained afterwards was compared to the control value using the paired t-test.
70
TIME ( minutes)
Figure 5* Effect of Na.B-OHB infusion on ammonia excretion
Values are niean+/- S.E.M. for 9 acidemic subjects. 3 successive measurements were obtained du ing steady water diuresis before Na.B-OHB infusion. These were averaged and represented ammonia excretion at time 0 (control value). Na.B-OHB wa, started at time 0. Each value obtained afterwards was compared to the control value using the paired t-test. Ammonia excretion following NaHC03 infusion in 3 subjects is also shown. The decline in ammonia excretion was significantly ■greater with Na.B-OHB than with NaHC03.
m in
TIME (minutes) A _ • - ---——
figure • Effect of Na.B-UHB. on sodium and chloride excretic
Values are mean+/~ V.E. . for 9 acidemic subjects.3 successive measurements were obtained during steady water diuresis before ba.B-OHB infusion.These were averaged and represented excretion values at time 0 (control value). Na.E-GEE was started, at time 0. Each value obtined afterwai was compared to the control value using the paired t-test. Sodium and chloride excretion fell significantly’ during Na.B-GHB infusion.
72
figure 7. Effect of NaliC03 infusion on sodium and chloride excretion.
Values are mean*/- S.E.M. for 3 acidemic subjects. 3 successive measurements were obtained during steady diuresis before I\aH.G03 infusion. These were averaged and represented excretion value at time 0 (control value). NaHC03 infusion was started at time 0. Each value obtained afterwards was compared to the control value using the paired t-test. Sodium and chloride excretion fell during GartG03 infusion.
^eq./
min
73
Na.Lactate infusion
Figure 8. Effect of Ea.Lact* on urinary ammonia excretion.
Values are meant-/- S.E.M. for 4 aeidemic subjects. 3 successive measurements were obtained during steady water diuresis before lactate infusion. These were averaged and represented ammonia excretion value at time 0 (control value). Lactate infusion was started at time 0. Each value obtained afterwards was compared to the control value using the paired t-test. Urinary ammonia excretion foil-owing NaHC03 infusion in 3 subjects is also shown. The decline in ammonia excretion was significantly greater with Na.Lact. than with Ea.IIC03e
74
TIME ( m inutes )
Figure 9. Effect of Na.Lactate on sodium and chloride excretion.
Values 'are mean+/-S.L.l;i. for 4 acedemic subjects. 3 successive measurements were obtained during steady water diuresis before Na.Lactate infusion. These were averaged and represented excretion value at time 0 (control value). Na.Lactate infusion was started at time 0. Each value obtained afterwards was compared to the control value using the paired t-test. Sodium and chloride excretion fell during Na.Lactate infusion
75
TUBULAR LUMEN RENAL TUBULAR CELL PERIRUBULAR BLOOD
1- glutaminase I; 2- glutamate dehydrogenase 3= phosphoenolpyruvate carboxykinase.
Figure 10. Factors affecting renal ammonia production and excretion.
76
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