Effect of ketone bodies on the renal excretion of ammonia

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Effect of ketone bodies on the renal excretion ofammoniaGary DesirYale University

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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

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a

3

day p

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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

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i—1 !-1

Effect

of

Na

3-O

HB

infusion

on

blo

od k

eto

ne

co

ncen

tration.

48

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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

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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

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i—I

m o X £ LO o CD 00 o -P co i—1 CO 00 i—1 CM CN

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U in i—1 00 CM N1 CO CM CO 0 i—1 in 1—1 CM z

ft 0

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53

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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

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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

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+ 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

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+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

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0 c o 0 0 0 0 a 0 0 0 G 0

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> £ co + 1 + 1 +1 +1 + 1 p 0 0 0 O

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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

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w £ /'“'I LO LD LO o in 0 0i 0 • ^ X p p

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rO

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CN cn

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w 0 0 o o CM i—1 1—1 -p 0 CO

| CTC

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CQ £ G G CQ

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11

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on

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, calc

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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

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s 0 G • 0 -H

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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

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12.

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CO

^

infu

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on

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a

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co

se,

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lucag

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, free

fatty

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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

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c •H 6

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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

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C O

G O

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4-1 r-| c 0 LD ro ro SP

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58

ro CM •M1 i—1 CO r- i—i CO

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13 0

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0 H

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CM i—1 0 i—1 •

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CM i—1 6 ro 13

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03 > 1-1 H Eh a 2 2

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co o ft CO ft CM > fd Li

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0 co 0 • • • • • • • aj u O

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0 p ft O co 04 1—1 1 1

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pH

, calc

ula

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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

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ft CO ft 0 ft G G G 0

0 O 0 E P 0 ft

ipH G ft 0 P 0

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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

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• • • • • • . 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

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TA

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16

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61

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63

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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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Lemieux, G., Vinay, P., and Cartier, P.: Characteris¬

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Effect of ketone bodies on the renal excretion of ammonia